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what are the Amino acid sequence?

Chapter 4 – Protein structure and function

Key points  

Protein structures (primary, secondary and tertiary), C­terminus and N­terminus, Protein folding and  conformation, Disulfide bonds formation, enzymes, substrate, antibodies structure, Enzyme functions, feedback  inhibition, positive and negative regulation, Protein modification and regulation, ATPase, GTPase, Amino acid sequence  

- A protein is assembled into a peptide chain by bonding individual amino acids together  using a covalent peptide bond

- The polypeptide chains are coded from a set of codons each with three letters signifying a  different amino acid

- Each type of protein has a unique amino acid sequence, which determines both its three dimensional shape and its biological activity.  Don't forget about the age old question of anu ziggurat

- There are 20 amino acids found in proteins – 10 non-polar (hydrophobic) and 10 polar  (hydrophilic)

what is Polypeptide chain?

- The amino acid side chains give the protein its unique properties. The side chain is not  involved in forming the peptide bond but it determined if the amino acid is polar, non polar, positively charged or negatively charged  

- Living cells contain an enormously diverse set of protein molecules, each made as a linear  chain of amino acids linked together by covalent peptide bonds.  

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Polypeptide chain

- The folded structure of a protein is stabilized by multiple noncovalent interactions between different parts of the polypeptide chain.  

- Formation of a peptide bond is a condensation reaction (a water molecule is eliminated) o a covalent peptide bond forms when the carbon atom of the carboxyl group of one  amino acid (such as glycine) shares electrons with the nitrogen atom from the  amino group of a second amino acid (such as alanine).  

what are the structure of protein?

- The longer the chain is, the more flexible it is  

- The shape of each of the folded proteins is constrained by sets of weak covalent bonds that  from within proteins.  

- Hydrogen bonds within the protein help stabilize its shape.  

- Hydrophobic (nonpolar) side chains tend to cluster inside the folded protein, they usually  hydrogen bond to other polar molecules or to the polypeptide backbone.  If you want to learn more check out joseph dixon rutgers

- Hydrophilic (polar) side chains tend to be near the outside of a folded protein and form  hydrogen bonds with water and other polar molecules.

- Chaperone proteins  

o Help in folding the protein by making the process more efficient and reliable  o Associations of these chaperons with the target protein requires an input of energy  from ATP hydrolysis.

o Some bind to the partly folded chains and help the fold along the most energetically favourable pathway.

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o Others form isolation chambers in which single polypeptide chains can fold  without risk of forming aggregates in the crowded conditions of the cytoplasm. C-terminus and N-terminus  We also discuss several other topics like reconstruction study guide

- A polypeptide backbone is formed from a repeating sequence of core atoms (NCC) known as the N-terminus and ends with a C-terminus.

- A protein is made of amino acids linked together into a polypeptide chain. The amino acids  are linked by peptide bonds to form a polypeptide backbone of repeating structure from  which the side chain of each amino acid projects.  

- Proteins are typically made up of chains of several hundred amino acids starting from the n terminus reading from left to right  

- The polypeptide backbone is formed from a repeating sequence of core atons (-N-C-C-)  found in every amino acid  

- The peptide chain has directionality because the two ends of each amino acid are  chemically different –  

o one has an amino group (NH3+) the other has a carboxyl group (COO-)  

o the end carrying the amino group is called the amino terminus, or N terminus, and the end carrying the free carboxyl group is the carboxyl  terminus, or C-terminus.  

Protein folding and conformation  

- The backbone model  

o shows the overall organization of the polypeptide chain and provides a straightforward way  to compare the structures of related proteins.  

- Ribbon model  

o Shows the emphasizes the various folds within the Polypeptide backbone  

- The wire model If you want to learn more check out sponch elements

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o Shows all the amino acid side chains. Useful in redicting which amino acids might be  involved in the proteins activity

- The space-filling model  

o Has a contour map which reveals which amino acids are exposed on the surface and shows  how the protein might look to a small molecule such as water or to another macromolecule  in the cell.  

- hydrophobic forces help proteins fold into compact conformations. polar amino  acid side chains tend to be displayed on the outside of the folded protein, where they can  interact with water; the nonpolar amino acid side chains are buried on the inside to form a  highly packed hydrophobic core of atoms that are hidden from water.  

Protein structure  

- Primary structure

o Protein structures begin with its amino acid sequence

- Secondary structure

o Alpha helix and beta sheets form within certain segments of the protein; these folds are  elements of the secondary structure.  

- Tertiary structure  

o The alpha helices beta sheets and any other loops and fold that form between n-terminal  and c-terminal  

- Quaternary structure

o If the protein molecule is formed as a complex of more than one polypeptide chain Protein family

- Members of a protein family contain similar amino acid sequences and have similar  structures.  

- Protein subunits  If you want to learn more check out econ 2113 ecu

o Dimer

o tetramer

- Once a protein has evolved a sufficiently stable conformation with useful properties, its  structure can be modified to in order to perform its designated specific functions  o Many present-day proteins can be grouped into protein families in which each  member has an amino acid sequence and a three-dimensional conformation that  closely resembles those of the other family members  

disulfide bond formation  

- Covalent bonds in the cell

- Doesn’t change the structure of the protein but it stabilizes the structure of the protein  - They are formed between sulfurs: you need a cysteine side chain because they form  between them  

- Requires oxidizing environment, because of this it is usually formed in the ER.  o The cytosol has a reducing environment and so these disulfide bonds cannot form in the cytosol  

o are formed before a protein is secreted by an enzyme in endoplasmic reticulum that links together two –SH groups from cysteine side chains that are adjacent in the  folded protein  If you want to learn more check out uw chem 237

- Covalent bonds between sulfurs in cysteine side chains

- Requires oxidizing environment

- Stabilizes proteins that are secreted

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o Disulfide bonds do not change a protein’s conformation, but instead act as a sort of  “atomic staple” to reinforce the protein’s most favored conformation.  

Alpha helix and Beta sheets

- Hydrogen bonds between neighboring regions of the polypeptide backbone often give rise  to regular folding patterns, known as α helices and β sheets.  

- These result from hydrogen bonds that form between the N-H and C=O groups in the  polypeptide bonds

- A-helix is generated when a single polypeptide chain turns around itself to form a  structurally rigid cylinder. A hydrogen bond is made between every fourth amino acid,  linking the C=O of one peptide bond to the N-H bond of another.

o Typically composed largely of amino acids with nonpolar side chains.  

o The polypeptide backbone, which is hydrophilic, is hydrogen-bonded to itself in the  α helix conformation, and it is shielded from the hydrophobic lipid environment of  the membrane by its protruding nonpolar side chains  

o Helix is a regular structure that is generated by placing many similar subunits next  to one another. A helix can be left or right-handed.  

- Alpha helix conformation – coiled-coil

o If multiple a-helices are wrapped around each other they form a stable structure  called a coiled-coil.  

o It forms when two or more helices have most of their non-polar side chains on one  side so that they can twist around each other with these side chains facing inwards  to minimize their contact with the aqueous environment  

- Beta sheets

o When neighboring segments are in the same direction the structure is called a  parallel beta sheet

o When the segments are in opposite directions the structure is called an antiparallel  beta sheet

o Several segments of an individual polypeptide chain are held together by hydrogen  binding between peptide bonds in adjacent strands

o The amino acid side chains in each strand project alternatively above and below the plane of the sheet

ALPHA HELICES BETA SHEETS

SHAPES AND  BONDS

- Helix is a regular structure that is generated by placing many similar subunits next to one  another. A helix can be left or right-handed.  Handedness is not affected by turning the  

- Amino acid side chains are not involved in forming  hydrogen bonds as such Alpha helix and beta  sheets and because they result from hydrogen bonds that form between the N-H and C=O groups in the

helix upside down, but it is reversed if he  

helix is reflected in a mirror.  

FORMATION - A-helix is generated when a single polypeptide chain turns around itself to form a structurally

rigid cylinder. A hydrogen bond is made  

between every fourth amino acid, linking the  

C=O of one peptide bond to the N-H bond of  

another.

COMPOSITION - Typically composed largely of amino acids with  nonpolar side chains. The polypeptide back

bone, which is hydrophilic, is hydrogen

bonded to itself in the α helix, and it is  

shielded from the hydrophobic lipid  

environment of the mem- brane by its  

protruding nonpolar side chains  

STRUCTURE - If multiple a-helices are wrapped around each  other they form a stable structure called a  

coiled-coil. Forms when a helices have most  

of their non-polar side chans on one side so  

that they can twist around each other with  

these side chains facing inwards

domains of a protein  

- Domains

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polypeptide bonds. They are very common, and the  protein chain adopts a regular, repeating form.  - Beta Sheets are made when hydrogen bonds form  between segments of a polypeptide chain that lies  side by side.

- Permit the formation fo amyloid fibers – insoluble  protein aggregares thar include those associated with neurodegenerative disorders  

- When neighboring segments are in the same direction  the structure is called a parallel beta sheet

- When the segments are in opposite directions the  structure is called an antiparallel beta sheet

o Any segment of a polypeptide chain that can fold independently into a compact,  stable structure.  

o The structure of many proteins can be subdivided into smaller globu- lar regions of  compact three-dimensional structure, known as protein domains.  

o A domain contains 40-350 amino acids and is folded into alpha helices and beta  sheets  

o It is a molecular unit from which larger proteins are constructed  

- proteins made up of several dozens of domains are intrinsically disordered  sequences  

o lack of folded structure makes them targets for the proteolytic enzymes that are  released when cells are fractionated  

o they can flex and bend which allows them to wrap arounf one or more target  proteins  

o bind with a high specificity and a low affinity  

o form flexible tethers between compact domains in a proteins to provide flexibility  while increasing the frequency of encounters between the domains.  

o Help scaffold proteins together by brining proteins together in an intracellular  signaling pathway  

o Main functions  

 Tethering interacting proteins  

 Tethering domains within a protein  

 Binding to a protein  

Subunits of a protein

- Weak noncovalent interactions allow polypeptide chains to fold into a specific  conformation  

- These interactions also allow proteins to bind to each other to produce larger structures in  the cell  

- A protein can contain binding sites that bind to the surface of a second protein  o The binding of two folded polypeptide chains at this site will create a larger protein,  whose quaternary structure has a precisely defined geometry.

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o Each polypeptide chain in such a protein is called

a subunit, and each subunit may contain more than

one domain.  

- Two identical folded polypeptide chains - (A)A protein 

with just one binding site can form a dimer with an identical 

protein  

o form a symmetrical complex called a dimer  

o the dimer is held together by interactions between two

identical binding sites  

- a symmetrical assembly of two different subunits -  

(B)Identical proteins with two different binding sites form a 

long helical filament  

o example: hemoglobin contains two copies of α-globin

and two copies of β-globin. each of these four

polypeptide chains contains a heme molecule, where

oxygen (O2) is bound.  

- (C)If the two binding sites are disposed appropriately in 

relation to each other, the protein subunits will form a closed 

ring instead of a helix  

- Actin filament is composed od identical protein subunits  

o The helical array of actin molecules in a filament contains thousands of molecules o Forms one of the filament systems in the cytoskeleton  

- Identical protein subunits can assemble into a complex  

Ligands

- The biological function of a protein depends on the detailed chemical properties of its  surface and how it binds to other molecules, called ligands.  

- Any substance that is bound by a protein (ion, small molecule, or a  macromolecule, is a ligand  

- Form noncovalent weak interactions (hydrogen bonds, electrostatic interactions, and van  der Waals attractions, and favorable hydrophobic forces) with proteins at the binding sites  - These interactions are WEAK transient interactions  

o They form weak noncovelent bonds so that effective binding requires many bonds  to be formed simultaneously  

o This is only possible is the surface contours of the ligand fit very closely with the  gap in the enzyme  

- As a result of the transient interactions - A protein can bind to a ligand by forming  transient (weak) interactions (which are noncovalent bonds) at the binding site of the  protein.

- The binding site Is where the substrate will bind to a proteins.  o Consists of a cavity in the protein surface formed by a particular arrangement of  amino acid side chains.  

o These side chains can belong to amino acids that are widely separated on the linear polypeptide chain but are brought together when the protein folds.  

- When molecules have poorly matching surfaces, few noncovalent interactions  occur = prevents incorrect and unwanted associations  

- Although the atoms buried in the interior of a protein have no direct contact with the  ligand, they provide an essential scaffold that gives the surface its contours and chemical  properties. Even tiny changes to the amino acids in the interior of a protein can change  the protein’s three- dimensional shape and destroy its function.

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Enzymes

- Enzymes bind to one or more ligands, called substrates, and

convert them into chemically modified products  

- enzymes act as catalysts that permit cells to make or break

covalent bonds  

o speed up reactions without being changed  

- can speed up reactions by  

o holding reacting substances together in a precise

alignment  

 binds two substrate molecules and orients

them to encourage a reaction between them

o rearranging the distribution og charge in a reaction

intermediate  

 binding of a substrate to enzyme rearranges

electrons in the substrate creating a partial

positive and partial negative charges that

favour a reaction  

o altering bond angles in the substrate to increase the rate of a particular reaction   enzyme strains the bound substrate forcing it toward a transition state to  favor a reaction  

- At the active site of an enzyme, the amino acid side chains of the folded protein are  precisely positioned so that they favor the formation of the high-energy transition states  that the substrates must pass through to be converted to product.  

- The energy barrier called activation energy must be overcome for the reaction to occur.  o So enzymes help in a variety of ways to lower the activation energy so the reaction  can happen and also make the reaction more energetically favourable Substrate Ligand

- The three-dimensional structure of many proteins has evolved so that the binding of a  small ligand can induce a significant change in protein shape.  

Substrate

- When a protein catalyzes the formation or breakage of a specific covalent bond in a ligand, the protein is called an enzyme and the ligand is called a substrate.  

Antibody structure  

- antibodies are immunoglobulin proteins produced by the immune system in  response to foreign molecules

o each antibody binds to a particular target molecule to either inactivate the target  directly or mark it for destruction  

- an antibody recognizes its target molecule, called an antigen

o antibodies are Y shaped with two identical antigen-binding sites  

o The antigen binding site is the most variable region of the antibody structure  - The antibody is composed of 4 polypeptide chains held together by disulfide bonds  o Two identical heavy chains  

 Has a variable domain close to the binding site,  

 And the region that is away from the binding site is the constant variable  domain  

o Two identical and smaller light chains  

 The light chain has the variable light chain (closer to the variable region) and  the constant light chain (further from the variable region)

o Each chain is made up of several similar domains  

- The antigen-binding site is formed where a heavy-chain variable domain and a light-chain  variable domain come close together

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o These two domains differ the most in their amino acid sequence in different  antibodies  

- The binding sites allow the antibody protein to interact with specific antigens o The folding of the polypeptide chain creates a cavity on the folded proteins suface,  where specific amino acid side chains are brought together in such a way that they  can form a set of noncovalent bonds in with certain ligands  

- A light chain that is closest to the antigen extends as a loop at one end of the variable  domain to form half of one antigen binding site of the antibody molecule  

The variable domain Is the part that is specific to different antibodies and is more antigen specific compared to the

enzyme functions

ENZYME FUNCTION

HYDROLASE General term for enzymes that catalyze a hydrolytic cleavage reaction  NUCLEASE Breaks down nucleic acids by hydrolyzing peptide bonds between amino acids PROTEASE Breaks down proteins by hydrolyzing peptide bonds between amino acids LIGASE Joins two molecules together; DNA ligase joins two strands together end­end ISOMERASE Catalyzes the rearrangement of bonds within a single molecule

POLYMERASE  Catalyzes the addition of phosphate groups to molecules. Protein kinases are an important  group of kinases that attach phosphate groups to proteins

PHOSPHATAS E

OXIDO

REDUCTASE

Catalyzes the hydrolytic removal of a phosphate group from a molecule

General name for enzymes that catalyze reactions in which one molecule is oxidized while the  other is reduced. Enzymes of this type are often called oxidases, reductases, or  dehydrogenases

ATPASE Hydrolyzes ATP. Many proteins have an energy­harnessing ATPase activity as part of their  function, including motor proteins such as myosin and membrane transport proteins such as  the sodium pump

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LYSOZYME releasing nutrients from ingested food particles and breaking down unwanted molecules for  recycling ot excretion from cell.

Provide safe environment for a vairiety of reactions involving hydrogen peroxide is used to  

PEROXISOMES

ALLOSTERIC  ENZYMES

inactivate toxic molecules

- have an Active site

o Where substrate binds

- Regulatory site  

o Where a regulatory bonding molecule binds  

- When a regulatory molecule binds to the regulatory molecule site, the  enzyme conformation will change which results in activation or deactivation of the enzyme.  

GTPASE - GTP binding proteins – these proteins usually act as switches that tu - GTP binding proteins will bind to a GTP molecule  

o Once it has the GTP it will perform a specific function  

o One form of GTP binding = movement of a specific receptors out of  

the nucleus so that when that receptor is bound to the GTP is moves  

out but when it goes out, the GTP will get hydrolyzed, it will remove  

one phosphate, then protein is bound to a GDP and it will be inactive

 When it is inactive and it is bound to GDP it will not have a  

high affinity for it, the GDP will be removed, and the protein is  

inactive again.

 For this to be activated the cycle will restart

ATPASE  Are generally found in motor proteins – found on cytoskeleton   The protein will bind to an ATP  

 once it binds to ATP = hydrolysis of the ATP will force to motor protein to  move.  

o Every time that the ATP becomes hydrolysed that motor protein will  

take one step

o So now you have ADP = ADP will be released, now the motor protein  

can take one more ATP, get it hydrolysed and move one more step  

further.  

Lysozyme  

- Catalyzes hydrolysis reactions by adding a molecule of water to a single bond between  two adjacent sugar groups in the polysaccharide chain causing the bond to break  - Reaction is energetically favourable because the free energy of the severed  polysaccharide chain is lower than the free energy of the intact chain.  

- For a colliding water molecule to break a bond, the polysaccharide molecule has to be  distorted into its transition state, in which the atoms aroung the bond have an altered  geometry and electron distribution  

o To distort the polysaccha- ride in this way requires a large input of energy from random molecular  collisions.  

- The active site (binding site) on lysozyme is where the catalysis of the chemical hydrolysis  reaction takes place.  

o When the enzyme-substrate complex forms, the enzyme cuts the polysaccharide  chain by catalyzing the addition of a water molecule to one of the sugar-sugar  bonds

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o The severed chain is then released, freeing the enzyme for further cycles of  cleavage  

- Overview

o A covalent bond in a polysaccharide is bent and then broken with the help of the  lysozyme catalyst  

o The catalyst alters the native shape of the molecule into its transition (higher  energy = less activation energy needed for reaction to proceed) then catalyzes the  addition of a water molecule to the sugar-sugar bond causing the bond to be  cleaved  

Feedback inhibition  

- Regulates the flow through biosynthetic pathways  

- Feedback inhibition  

o regulates connected metabolic pathways  

o triggers a conformational change in an enzyme  

- The cell controls enzymatic activities by confining sets of enzymes to particular subcellular compartments or by switching proteins on/off

- for example, an enzyme acting early in a reaction pathway is inhibited by a late product of that pathway. Thus, whenever large quan- tities of the final product begin to accumulate,  the product binds to an earlier enzyme and slows down its catalytic action, limiting further  entry of substrates into that reaction pathway

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positive and negative regulation

- Feedback inhibition is also negative regulation – it prevents an enzyme from acting  - Enzymes can also be subject to positive regulation in which the enzymes activities are  stimulated by a regulatory molecule rather than being supressed. This occurs when a  product in one branch of the pathway stimulates the activity of an enzyme in another  pathway  

- The activities of most enzymes within the cell are strictly regulated. One of the most  common forms of regulation is feedback inhibition, in which an enzyme early in a  metabolic pathway is inhibited by the binding of one of the pathway’s end products.  allosteric enzymes

- Most enzymes are allosteric proteins that can exist in two conformations that  differ in catalytic activity, and the enzyme can be turned on or off by ligands  that bind to a distinct regulatory site to stabilize either the active or the  inactive conformation.  

- Can adopt two or more slightly different conformations and their activity can be regulated  by a shift from one to another  

- Each protein conformation will have slightly different contours on the surface thus the  proteins binding site for specific ligands will be altered when the enzyme undergoes a  conformational change  

- Each ligands will stabilize the conformation that is binds to most strongly and at high  enough concentrations, a ligand will switch the population of proteins to the conformation  it prefers  

- Phosphorylation causes a conformational change  

o When a phosphate group is attached to one or more of an enzymes side chains it  will cause a conformational change  

o The addition of a phosphate group to a side chain is catalyzed by protein kinase and is reversed by protein phosphatase  

o This conformational change can in turn affect the binding of ligands elsewhere on  the protein surface = altering the proteins activity  

- The addition and removal of phosphate groups from specific proteins occurs in response to signals that specify some change in the cells state  

- Phosphorylation can either stimulate protein activity or inhibit it, depending on the protein  involved and the site of phosphorylation.  

- Highly efficient protein machines are formed by assemblies of allos- teric proteins in which the various conformational changes are coordinated to perform complex functions.  -

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protein modification and regulation  

- The activities of most enzymes within the cell are strictly regulated. One of the most  common forms of regulation is feedback inhibition, in which an enzyme early in a  metabolic pathway is inhibited by the binding of one of the pathway’s end products.  

- Covalent modifications added to a protein’s amino acid side chains can control the location and function of the protein and can serve as docking sites for other proteins.  

- Many thousands of proteins in a typical eukaryotic cell are regulated by cycles of  phosphorylation and dephosphorylation.  

Enzyme activity – protein degradation  

• Proteolysis is the process of breaking down the protein to amino acid

– Proteases

– an mirNa targets a complementary mrNa molecule for destruction. 

Takes place when a protein has been marked by a 

polyubiquitinate chain and is going to be directed to a  proteosome. 

Within the proteosome the polyubiquitin is going to get recycled  and removed form the protein an dpolypeptide

The polypeptide is going to be lineralized  ad is going to go  through the central cylinder that is made of proteases.

Proteases will the brak down the polypeptide into amino acdids  And the ami

Takes place in the lysosome

1.addition of polyubiquitin chain tag the protein  and mark it for degradation 

2. go through the proteosome itself

3.it will be linearized

4.the central cylinder is made of proteases 5. once it goes through the  central cylinder it is  broken down 

6.the amino acids are then released as individuals  to be recycled and used again

– each precursor mirNa transcript is processed to form a double­ stranded

intermediate, which is further processed to form a mature, single

stranded mirNa. this mirNa assembles with a set of proteins into a

complex called rISc, which then searches for mrNas that have a

nucleotide sequence complementary

to its bound mirNa. Depending on how extensive the region of complementarity is, the target 

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mrNa is either rapidly degraded by a nuclease within the rISc or transferred to an area of the  cytoplasm where other cellular nucleases destroy it. 

ATPase

- Motor proteins produce directed movement in eukaryotic cells through conformational  changes linked to the hydrolysis of ATP to ADP.  

- Are generally found in motor proteins – found on cytoskeleton  

- The protein will bind to an ATP  

- once it binds to ATP = hydrolysis of the ATP will force to motor protein to move.  o Every time that the ATP becomes hydrolysed that motor protein will take one step - So now you have ADP = ADP will be released, now the motor protein can take one more  ATP, get it hydrolysed and move one more step further.

GTPase

- b

- Once it has the GTP it will perform a specific function  

o One form of GTP binding = movement of a specific receptors out of the nucleus so  that when that receptor is bound to the GTP is moves out but when it goes out, the  GTP will get hydrolyzed, it will remove one phosphate, then protein is bound to a  GDP and it will be inactive

- When it is inactive and it is bound to GDP it will not have a high affinity for it, the GDP will  be removed, and the protein is inactive again.

- GTP-binding proteins also regulate protein function in eukaryotes; they act as molecular  switches that are active when GTP is bound and inactive when GDP is bound; turning  themselves off by hydrolyzing their bound GTP to GDP.  

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- GTP binding proteins – these proteins usually act as switches that tu

- GTP binding proteins will bind to a GTP molecule  

o Once it has the GTP it will perform a specific function  

o One form of GTP binding = movement of a specific receptors out of the nucleus so  that when that receptor is bound to the GTP is moves out but when it goes out, the  GTP will get hydrolyzed, it will remove one phosphate, then protein is bound to a  GDP and it will be inactive

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 When it is inactive and it is bound to GDP it will not have a high affinity for it,  the GDP will be removed, and the protein is inactive again.

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Chapter 11 – Membrane structure  

Key points  

Glycocalyx, Peripheral and integral proteins, Scramblase and Flipase, Glycolipid formation, Lipid Synthesis,  function of Cholesterol, Saturated vs Unsaturated lipids, liposome,

Amphipathic molecules  

- Amphipathic molecules have both a

hydrophilic and a hydrophobic end.  

o This property plays a crucial part in

driving these lipid molecules to

assemble into bilayers in an

aqueous environment.  

- Membrane lipid molecules are

amphipathic, having both hydrophobic

and hydrophilic regions. This property

promotes their spontaneous assembly

into bilayers when placed in water,

forming closed com- partments that

reseal if torn.  

- hydrophilic molecules dissolve readily

in water because they contain either

charged groups or uncharged polar

groups that can form either electrostatic

attractions or hydrogen bonds with water molecules  

- Hydrophobic molecules are insoluble in water because almost all of their atoms are  uncharged and nonpolar; they therefore cannot form favorable interactions with water  molecules. Instead, they force adjacent water molecules to reorganize into a cagelike  structure around them  

- This structure is more highly ordered than the rest of water so its formation requires free energy  

- This energy cost is minimized when the hydrophobic molecules cluster together  (limits contact with the surrounding water molecules) which is why they cluster on  the inside of the membrane and don’t let water through  

- Consequently, the hydrophobic ends of amphipathic molecules tend to be exposed to air at  air-water interfaces, or, the interior of an aqueous solution, they will always cluster  together to minimize their contact with water molecules.

- Amphipathic molecules are subject to conflicting forces which is resolved by the  formation of the lipid bilayer which satisfies both regions of the lipid and is thus energetically favourable  

o This conformation also limits movement of molecules through the membrane by  forming a barrier to prevent movement of specific molecules through the bilayer.

-

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Phospholipids  

- A group of compounds like fatty acids that dissolve in organic solvents and fat. - Types of phospholipids depends on the different groups attached to the phosphate group  in phospholipid

o Are insoluble in water  

o Made of a hydrophobic/ lipophilic tail and a hydrophilic/lipophobic head.  o Forms the plasma membrane and organelle membrane  

- The nucleus, chloroplast, and mitochondria have a double membrane. Others only have  one lipid bilayer

- Are very important in transport in and out of the cell.

- The hydrophilic head can change to form different lipids

- The length of tail varies depending on the number of carbons.

Glycolipids  

- Oligosaccharide branch containing 14 sugars is added to proteins

- Have sugar as hydrophilic head attached to dolichol  

o The sugar group is transferred to Asn (amino acid) by oligosaccharide transferase - Glycoproteins are transferred to Golgi after synthesis for modification  - Formed by glycosylation. Typically found on the extracellular side of the membrane  - Protect the membrane from damage

Membrane bilayer

- The lipid bilayer is fluid, and individual lipid molecules are able to diffuse within  their own monolayer; they do not, however, spontaneously flip from one  monolayer to the other.  

- The two lipid monolayers of a cell membrane have different lipid compositions,  reflecting the different functions of the two faces of the membrane.  - There are three major classes of membrane lipid molecules: phos- pholipids,  sterols, and glycolipids.  

- Phospholipids are the most common lipids in the membrane. There are also glycolipids. o The phospholipid bilayer is formed because the hydrophilic polar head is attracted  to water and the phospholipid non-polar tail is repelled by the water and faces the  inside of the membrane.

o The hydrophobic tails fact each other and form an area that doesn’t allow water to  pass through. This is what give you a bilayer and not a water layer.  

- They are hydrophobic because they cannot form bonds with hydrogen in water. So  hydrogen bonds in water molecules will form stronger bonds and surround the  hydrophobic molecule.  

o The hydrophilic heads form hydrogen bonds with the water and therefore they have  hydrophilic properties.  

- Composition  

o Hydrocarbon tails define the fluidity of the membrane  

o Shorter tails result in higher fluidity = higher permeability = more molecules can  pass through  

o Unsaturated hydrocarbon tail decreases fluidity.  

Saturated and unsaturated  

- Saturated lacks a kink  

o Hydrocarbon tail with no double bond has a full complement of hydrogen atoms  - Phospholipid tails that have double bonds do not contain the maximum number of  hydrogen atoms that could, in principle, be attached to its carbon backbone. o These are unsaturated with respect to hydrogen

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o Unsaturated phospholipid molecules have a kink in one of the two  hydrophobic tails  

o Each double bond creates a small kink in the tail. The kink makes it more difficult for the tails to pack tightly against each other.  

o For this reason, lipid bilayers that contain a large proportion of unsaturated  hydrocarbon tails are more fluid than those with lower proportions  

o The kink forms a gap in the membrane which makes it more permeable - Cholesterol can fit between the gap formed by the kink to reduce flexibility and  permeability  

o Because cholesterol molecules are short and rigid, they fill the spaces between  neighboring phospholipid molecules left by the kinks in their unsaturated  hydrocarbon tails  

- At higher temperatures there are fewer double bonds  

Cholesterol  

- Cholesterol modulates cell membrane fluidity in animals cells  

- Cholesterol fits in the gaps formed by the unsaturated tails.  

- Less permeable, less flexible  

- As a result of the kink that happens when double bonds are present, there is a gap in  membrane which allows molecules to go through.  

- Cholesterol goes into those gaps to make it more rigid, contain itself, and restrict  movement through the membrane.  

Liposome  

- Are vesicles formed by phospholipids and used in the transport of drugs  - Molecules are added inside of liposomes and the liposome merges with target cell to  deliver drug.

- Tagging liposomes is don’t by different proteins.  

Lipids movement through the membrane  

- Lipids can move from one monolayer to another, with the help of enzymes.  - However, lipids can freely move horizontally through the bilayer without enzymes.

Lipid synthesis in ER

1. Using free fatty acids as substrates, the enzymes bound to the cytosolic monolayer/  surface of the ER deposit the newly made phospholipids in the cytosolic half of the ER  bilayer

2. Results in expansion of monolayer side facing cytosol  

3. Scramblase enzymes catalyze the random transfer of phospholipids added to cytosolic  face and moves them to the side facing ER movement – allowing the membrane to grow  as a bilayer

a. Result: creating symmetric growth of both halved of bilayer  

b. Transport is random which enhances symmetric growth  

4. Newly synthesized lipids leave the ER and are transported to golgi apparatus  5. Flippases in the Golgi specifically remove certain phospholipids from the noncytosolic  monolayer and flip them to the cytosolic side (not glycolipids)

6. Glycolipids are composed of sugar and lipids get sugar groups from golgi to become  glycolipids.

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Most cells are asymmetric. Most are circular so you have more phospholipids on outer layer than the inner  layer. This is why growth is asymmetric.

-

Flippase  

- Flippases in the Golgi specifically remove phosphatidylserine and  

phosphatidylethanolamine from the noncytosolic monolayer and flip them to the cytosolic  side  

o The monolayer facing lumen typically have less lipids

o This results in asymmetry of the phospholipid membrane and may help subsequent  vesicle budding = different monolayers have different lipids  

o DON’T MOVE GLYCOLIPIDS

- Moves specific lipids from one monolayer to another.

o It provides asymmetry in membrane.  

- Different membranes require different compositions so flippase moves specific  phospholipids depending on what specific structure is needed.  

- Glycolipids remain where they are formed and they need to be on the extracellular space because of they need to take sugar group from the Golgi

Scramblase

- Remove randomly selected phospholipids from one half of the lipid bilayer and insert them in the other half.  

- As a result the newly made phospholipids are redistributed equally between each  monolayer of the ER membrane  

- Some of this newly assembled membrane will remain in the ER;  

- the rest will be used to supply fresh membrane to other compartments in the cell.  - Bits of membrane are continually pinching off the ER to form small, spherical vesicles that  then fuse with other membranes, such as those of the Golgi apparatus.

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- Additional vesicles bubble from the Golgi to become incorporated into the plasma  membrane.  

Vesicle transport

- Cell membranes have distinct inside and outside faces: cytosolic monolayer always faces  the cytosol  

- The noncytosolic monolayer always faces the cell exterior

- Originate in ER, when the detach from ER they translocate to Golgi  

- Then they translocate to plasma membrane where they are then released from the cell.  - Transport vesicles dock to organelles to transfer cargo molecules  

- Process with high specificity  

- Vesicles fuse to target membrane.

glycolipids

- Located mainly on the plasma membrane and only in the noncytosolic half of the bilayer  - Their sugar groups face the cell exterior where they form part of a continuous coat of  carbohydrate that surrounds and protects animal cells.  

- Glycolipid molecules acquire their sugar groups in the Golgi apparatus where enzymes  that cause this chemical modification are confined.  

- These enzymes are oriented such that the sugars are added only to lipid molecules in the  noncytosolic half of the membrane bilayer  

- Once a glycolipid molecule has been created in this way it remains trapped in this  monolayer as there are no flippases that transfer glycolipids to the cytosolic side.  - Thus when a glycolipid molecule is finally derived to the plasma membrane, it displays its  sugars to the exterior of the cell  

Types of membrane proteins

- Membrane proteins are responsible for most of the functions of cell membranes, including  the transport of small, water-soluble molecules across the lipid bilayer.  

FUNCTIONAL CLASS PROTEIN EXAMPLE SPECIFIC FUNCTION 

TRANSPORTERS  Na+ pump Actively pump charged ions out of cells and  K+  ions in

ION CHANNELS  K+ leak channel  Allows K+ ions to leave cells, therby having a  major influence on cell excitability 

ANCHORS  Integrins  Link intracellular actin filaments to extracellular  matric proteins 

RECEPTORS  PDGF receptor Binds extracellular PDGF to generate intracellular  signals that cause the cell to grow and divide

ENZYMES Adenylyl cyclase Catalyzes the production of the small intracellular  signaling molecule cyclic AMP in response to 

extracellular signals 

FUNCTIONAL CLASS EXAMPLES  STRUCTURE 

TRANSMEMBRANE  PROTEIN 

MONOLAYER 

ASSOCIATED A­HELIX

Bacteriorhodopsin (proton  pump)

Extend across the bilayer as a single alpha  helix, as multiple alpha helices or as a  rolled up betta barrel 

Anchored to the cytosolic half of the lipid  bilayer by an amphipathic alpha helix

LIPID LINKED Linked to either side of the bilayer by a 

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covalently attached lipid molecule  

PROTEIN ATTACHED  Attached to the membrane by relatively  weak noncovalent interactions with other 

membrane proteins

Integral proteins  

- Proteins that are attached to the lipid bilayer can be removed by disrupting the bilayer  detergents  

o Transmembrane protein  

o Monolayer a-helix  

o Lipid-linked  

Peripheral proteins  

- Can be released from the membrane by more gentle extraction procedures that interfere  with protein-protein interactions but leave the lipid bilayer intact  

- Indirectly attached to bilayer through attaching to proteins connected to lipids  o Protein-attached  

Alpha helices in transmembrane proteins  

- Other membrane proteins do not extend across the lipid bilayer but are  attached to one or the other side of the membrane, either by noncovalent  association with other membrane proteins, by covalent attachment of lipids, or  by association of an exposed amphipathic α helix with a single lipid monolayer.  

- Transmembrane hydrophilic pore can be formed by multiple amphipathic alpha helices.  o Peptide bonds that join the successive amino acids in a protein are polar, making  the polypeptide backbone hydrophilic  

o Because water is absent from the interior of the bilayer, atoms forming the  backbone are driven to form hydrogen bonds with one another.  

o Hydrogen bonding is maximized If the polypeptide chain forms a regular alpha helix  - In the membrane spanning alpha helices

o The hydrophobic side chains are exposed on the outside of the helix, where they  can contact the hydrophobic tails, while atoms in the polypeptide backbone form  hydrogen bonds with one another on the inside of the helix  

- single pass transmembrane proteins  

o cross the membrane only once  

o many are receptors for extracellular signals  

- multipass transmembrane proteins  

o one or more of the membranes spanning region are amphipathic – cross the lipid  bilayer multiple times  

 formed from alpha helices that contain both hydrophobic and hydrophilic  amino acid side chains  

o amino acids are arranged so that the hydrophilic side chains are concentrated on  the outside of the helix

o while hydrophobic side chains are on the inside of the helix  

o In the hydrophobic environment of the lipid bilayer, alpha helices of this sort pack  side by side in a ring, with the hydrophobic side chains forming the lining of the  hydrophilic pore through the lipid bilayer  

o Function in the selective transport of small, water soluble molecules,  especially inorganic ions  

- A transmembrane polypeptide chain usually crosses the lipid bilayer as an alpha helix.

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o An alpha helix containing about 20 amino acids is required to completely transverse the cell membrane

Beta sheets in transmembrane proteins  

Beta barrel

- Transmembrane proteins extend across the lipid bilayer, usually as one or more  α helices but sometimes as a β sheet rolled into the form of a barrel.  - Amino acid side chains that face the inside of the barrel (line the aqueous channel) are  hydrophilic  

- The amino acid side chains on the outside of the barrel are hydrophobic  o Contact the hydrophobic core of the lipid bilayer  

- Example: the porin protein  

o Form large water filled pores in mitochondrial and bacterial outer membranes  o Mitochondria and some bacteria are surrounded by a double membrane and porins  allow the passage of small nutrients, metabolites, and inorganic ions across their  outer membranes while preventing unwanted larger molecules from crossing

Glycocalyx  

- Many of the proteins and some of the lipids exposed on the surface of cells have attached sugar chains, which form a carbohydrate layer that helps protect and  lubricate the cell surface, while also being involved in specific cell–cell  recognition.  

- The cell surface is coated with carbohydrate.  

- Some lipids on the outer layer of the plasma membrane have sugars covalently attached  to them.  

- Glycoproteins  

o Have short chain sugars called oligosaccharides linked to them  

- Proteoglycans  

o Contain one or more long polysaccharide chains  

- All of the carbohydrate on the glycoproteins, proteoglycans and glycolipids is located on  the outside of the plasma membrane, where it forms a sugar coating called the glycocalyx o Carbohydrates on the outer membrane form glycocalyx  

- Glycocalyx is carbohydrate surface that covers and protects the plasma membrane from  environmental hazards.  

o The carbohydrate layer is made of oligosaccharide side chains attached to the  membrane glycolipids, glycoproteins, and the polysaccharide chains on membrane  proteoglycans  

o Glycoproteins that have been secreted by the cell and then adsorbed back onto its  surface can also contribute  

- All the carbohydrate is on the external (noncytosolic) surface of the plasma membrane  - Cell surface carbohydrates  

o Protect and lubricate the cell  

o They also have an important role in cell-cell recognition and adhesion  o They recognize a particular site on a another protein (proteins called lectins are  specialized to bind to particular oligosaccharide side chains) the side chains are  diverse.  

- Sugars can be joined together in a linear chain by a variety of covalent linkages to form  elaborate branched structures

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Chapter 12 – transport across cell

membranes  

Key points  

Facilitated transport, Na Pump, Ouabain, Ca Pump, how ions can be transported, Aquaporin, H pump function, 

Simple diffusion  

- Which molecules can or cannot diffuse  

- Small non-polar molecules diffuse through the membrane but diffusion is very slow - Dissolve readily in lipid bilayer and therefore diffuse rapidly across them.  - Cells depend on this permeability to gases for the cell respiration processes  - Uncharged small polar molecules also diffuse readily across membrane but the diffusion is  slow.

- Larger uncharged polar molecules barely diffuse without help  

- Occurs spontaneously  

- Channel proteins form pores across lipid bilayer through which solutes can passively  diffuse  

Facilitated transport  

- Requires the input of energy as ATP

- To move a solute against its concentration gradient, a membrane transport protein must  do work that thus energy is needed.

- Is carried out by special types of transporters called pumps which harness an energy  source to power the transport process

membrane permeability  

- Lipid bilayer of cell membranes is 

- Highly permeable to small nonpolar molecules  

- Oxygen, carbon dioxide

- Less to small polar molecules  

- water

- highly impermeable to large water-soluble molecules  

- ions

- transfer of nutrients, metabolites, and inorganic ions across cell membrane depends on membrane  transport proteins

membrane potential  

- Determined by the unequal distribution of charged ions on the two sides of the membrane - It is altered when these ions flow through open ion channels in the membrane. - When two forces support movement of cations from outside to inside the membrane the  movement is fast.

- Concentration supports electric force doesn’t so movement will be slower than if both  supported.

- Electric forces  

- Charged molecules follow charged on either side of the membrane  

- Charged molecules  

- Net force of concentration gradient and charge = electrochemical gradient

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- Both forces supports movement of cations from outside  

- Negative value of the resting membrane potential  

- Depends mainly on the K+ gradient and the operation of K+ selective leak channels - At this resting potential, the driving force for the movement of K+ across the membrane is almost zero  

- Neurons propagate electrical impulses in the form of action potentials. Actional potentials  are mediated by the voltage-gated Na+ channels that open in response to depolarization  of the membrane  

- Voltage gated Ca 2+ channels in a nerve terminal couple the arrival of an action potential  to neurotransmitter release at a synapse.

ions inside and outside the cell  

- K

IONS INSIDE IONS OUTSIDE

K+ Na+ 

Mg++

Ca++ 

H+ H+

Cl

Channels  

- Not specific to one type. Any molecule with target size and charge will be transported  through.  

- Molecules transfer based on their size and charge

- Excitatory NTs open transmitter-gated cation channels that allow influx of Na+, which  depolarized the postsynaptic cells plasma membrane and encourages the cell to fire and  action potential.

- Inhibitory NTs open the transmitter-gated Cl- channels in the postsynaptic cell membrane  to make it harder for the membrane to depolarize.

-

Ion channel Typical location Function 

K+ leak channel Plasma membrane of most  animal cells

Maintenance of resting  membrane potential  

Voltage-gates Na+ channel  

Voltage-gated Ca2+ channel

Plasma membrane of nerve cell  axon

Plasma membrane of nerve  terminal  

Generation of action potential  

Stimulation of neurotransmitter  release  

Acetylcholine receptor Plasms membrane of muscle  cell

Excitatory synaptic signaling  

GABA receptor (Ligand gated ion channel) Glycine receptor (ligand gated ion channel)

Plasma membrane of many  neurons at synapses

Plasma membrane of many  neurons at synapses

Inhibitory synaptic signalling  Inhibitory synaptic signalling  

Mechanically-activated cation channel

Transmitter gated ion channels

Auditory hair cell in inner ear Detection of sound vibrations  

Synapse convert chemical signals back  into electrical on the  

postsynaptic target cell.

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Transporters  

- Transporters carry specific molecules across the membrane by undergoing conformational changes that  expose the solute­binding site first one side of the membrane and then on the other 

- Different organelles requires different types of transporters based on function and requirements  Transport

type 

H+

transported

lysosome Imports H+ to acidify the lysosome interior and other transporters that move digestion products out of the lysosome into the cytosol.

Transporters Plasma membrane Sugars, amino acids and nucleotides into cell Transporters Mitochondria Transport pyruvate that mitochondria uses a fuel for generating ATP as well as exporting ATP once its synthesized.  

Glucose

transporter  

Membrane transport

protein  

Ligand-gated channels

Plasma membrane Consists of a polypeptide chain that crosses the membrane 12 times  can adopt many confotmations. Because glucose is uncharged, the  

electrochemical gradient is zero. Thus the direction in which it Is  

transported is determined by its concentration gradient alone.  

Lipid membrane

Ca2+ pump Lipid membrane 

H+ Pump (ATPase)

Mitochondria

Ion channels Lipid membrane Allow inorganic ions of appropriate size and charge to cross the  membrane. Most are gated and open transiently in response to a  

specific stimulus  

K+ leak

channels

Na+ pump (ATPase)

Plasma membrane of animal cells  

Actively transports Na+ out of the cell and K+ into the cell to maintain  a steep Na+ gradient across the plasma membrane that is used to  drive other active transport processes and to convey electrical signals.

- Hydrogen pumps, pump hydrogen protons into lysosome which makes acidic environment  - ATP pumps move ATP out of mitochondria and ADP into mitochondria 

- Transporters 

- Molecule or protein must fit into its binding site  

- Transport is very specific to the molecules they are designed for.

Passive transport

- Passive transport can occur through channels or transporters.  

- Molecules can go through channels or diffuse through membrane based on only  concentration or electrochemical gradient

Passive transporters  

- An uncharged solute moves spontaneously down its concentration gradient  - Are specific to certain molecules. Specificity is so high that it will not take isomer of  specific molecule. Glucose binds to transporter and transporter will change conformation  and release glucose on its other binding site.

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- Break glycogen down to glucose as a result there is a higher concentration of glucose in  extracellular space compared to cytosol.

- Glucose can then bind to transporter and follow concentration gradient into cell.  - For the passive transport of a charged solute, its electrochemical gradient determines its  direction of movement, rather than its concentration alone.

- Both transporters and channels can mediate passive transport

Active transport  

-

Active transporters  

- Transporters can act as pumps to mediate active transport

- Solutes are moved uphill against their concentration or electrochemical gradients  - This process requires an input of energy that is provided by ATP hydrolysis, a downhill flow  of Na+ or H+ ions, or sunlight  

- Can only occur through transporters  

- Requires input of energy because active transport moved molecules against their  concentration gradient.  

-

Voltage gradient

- Most cell membranes have a voltage across them – a difference in charge referred to as a  membrane potential.  

- The membrane potential exerts a force of on any molecule that carries an electric charge. - The cytosolic side of the plasma membrane is usually at a negative potential relative to  the extracellular side, so the membrane potential tends to pull positively charged solutes  into the cell and drive negatively charges ones out.

-

Electrochemical gradient

- Electrochemical gradient when voltage and concentration gradient work in the same  direction  

- Positive ions attracted to negative charge on the other side of the membrane  - Concentration is lower on the other side of the membrane  

- Electrochemical gradient when voltage and concentration gradients work in opposite  directions  

- The gradient will follow the concentration gradient even if it goes against electrochemical  gradient

Pumps

- Transporters can act as pumps to mediate active transport,

- An active transport of solutes against their electrochemical gradient is essential to  maintain the appropriate intracellulat ionic composition of cells and to import solutes that  are at a lower concentration outside the cell than inside.  

- For these purposes cells depend on transmembrane pumps which can carry out active  transport in three main ways  

- ATP-driven pumps  

- Hydrolyse ATP to drive uphill transport  

- Coupled-pumps  

- Link the uphill transport of one solute across a membrane to the downhill transport of  another

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- Light-driven pumps  

- Found mostly in bacterial cells, use energy derived from sunlight to drive uphill transport

Osmosis

- Movement of water down its concentration gradient

- Water is uncharged so it only depends on the concentration gradient.

- A lot of movement of water into a cell that happens too fast and all at once causes  swelling and bursting vacuoles and cytoplasm  

- Thus the total concentration of solute particles inside the cell—also called its osmolarity— generally exceeds solute concentra- tion outside the cell. The resulting osmotic gradient  tends to “pull” water into the cell. This movement of water down its concentration  gradient— from an area of low solute concentration (high water concentration) to an area  of high solute concentration (low water concentration)—is called osmosis.

Osmolarity

- Total concentration of solute inside the cell

- Water flows to area with higher concentration of solute and lower concentration of water -

Aquaporin  

- Water molecules diffuse rapidly through aquaporin channels in the plasma membrane of  some cells  

- Each aquaporin channel forms a pore acress the bilayer, allowing the selective passage of  water molecules  

- An aquaporin tetramer is the biologically active form of aquaporin.

-

Ion channels

- Allow inorganic ions of appropriate size and charge to cross the membrane. - Most are gated and open transiently in response to a specific stimulus  - When when activated by a specific stimulus, ion channels do not remain continuously  open.  

- They flicker randomly between open and closed conformations.  

- An activating stimulus increases the proportion of time that the channel is in open state. - Ions can go through pumps

Na+ pumps  

- Na+ transport is coupled by K+ import

- Na binds to region on transporters and move with concentration gradient  - Will take phosphate, phosphorylate itself and move with concentration gradient.  - Inhibition of any steps in the Na+ pump halts the process completely.

- Ouabain is an inhibitor of this process.

- Ouabain blocks potassium from binding to pump through inhibition the transport of sodium  and potassium is inhibited.

1. ATP phosphorylates the pump  

2. The pump changes conformation  

3. The pump moves

a. 3 Na+ from cytosol  

b. 2 K+ from extracellular space to cytosol  

c. This maintains a high concentration of sodium in extracellular space  

d. And a high concentration of potassium in the cytosol

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- If any of these steps are inhibited, the whole process becomes inhibited 

-

Ca++ pumps  

- Movement is specific to calcium

- Sarcoplasmic reticulum only found in muscle cells 

- Contains pumps that move calcium from cytosol into lumen of sarcoplasmic reticulum - Uses ATP as a source of energy

- It is also an ATPase so it takes phosphate group from ATP will be attached to as aspartic acid and ATP is attached to pump.  

- Phosphorylation takes place on aspartic acid  

1. Calcium binds to binding site (calcium binding cavity)

2. Pump is phosphorylated by ADP

a. Which means there is an energy input  

3. The pump changes conformation  

4. There is movement of  

a. 2 Ca+ from cytosol to lumen of sarcoplasmic reticulum  

-

Coupled transport  

- Movement of one transported, down its gradient is liked to and provides energy for the transport of an ion - Symport = same direction  

- Antiport= opposite directions (one with one against concentration gradient) - One molecule forces the other to move against concentration gradient  

1. Molecule needs to move  

a. It follows its chemical gradient

2. The energy created by the chemical gradient is used to transport the other molecule.

Proton Pumps  

- Chemiosmotic generation of energy begins as NADH and FADH2 donate electrons to the electron  transport chain and start the oxidative phosphorylation process.

- Oxidative phosphorylation 

o The consumption of oxygen and phosphorylation of ADP  

- Production of pyruvate  transport pyruvate and fatty acids into mitochondria  oxidation of pyruvate into acetyl CoA  acetyl CoA  used by citric acid cycle to produce NADH and FADH2  NADH and  FADH2 donate their electrons to ETC  electrons enter ETC, ETC pumps protons into the inner  membrane space  creates a proton gradient which then goes through ATP synthase  when the proton  gradient goes through ATP synthase the energy produced is enough to phosphorylate ADP  ATP 

Electron carriers  

- Ubiquinone 

o Is a hydrophilic electron carrier found within the mitochondrial inner membrane  o It takes electrons from NADH dehydrogenase and passes the electrons to  cytochrome C reductase complex.

o Is the entry point for electrons donated by FADH2

 Redox potential goes from low to high in ETC

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 FADH2 gives one less ATP than NADH because eit enters the ETC one protein  later than NADH  

 FADH2 cannot donate electrons to NADH dehydrogenase complex  

because the tendency of FADH2 to hold onto its electrons is much  

higher than the tendency for NADH dehydrogenase to take electrons.  

o Quinone carries the same function during photosynthesis

- Cytochrome C 

o Is the final electron carrier in the respiratory chain  

o Takes electrons from the cytochrome C reductase complex

 Passes electron to cytochrome C oxidase complex  

o Removes electrons from cytochrome C  

 4e- + 4H+ + O2  2H2O

o Contains an oxygen binding site  

 Made up of a Heme group and copper atom  

 E- is passed to oxygen at binding site.  

 Oxygen binding site with the heme group and copper atom transfer electrons  and protons to oxygen because oxygen will accept electrons and protons at  the same time  

o If this doesn’t occur, oxygen will have a negative charge and will create an oxygen  radical (O2-) which is dangerous for the cell  

 Oxygen should be bound to binding site and accept the correct number of  ions to prevent formation of an oxygen radical.

 Radical is damaging to DNA and proteins  

Electron transport and proton pumping  

- High concentration of protons in the inter membrane space 

- Hydrogen atoms are the most abundant atom in living cells (OIL RIG)

o Reduced molecules acquire an electron and are neutralized by H donation from water (reduction  is gaining)

o Oxidized molecules lose electrons and are neutralized by donating H to water (Oxidation is  losing)

- Every time a reduction or oxidation occurs, the molecules will be neutralized by a proton  o Reduced molecule get a negative charge

o Oxidized molecules get a positive charge 

- Transient intermediate is negatively charged 

o Allows it to obtain a proton 

o Passes proton onto reduced electron carrier 

- Every time an electron is passed, the reduced protein will also gain an electron 

o When protein complex passes the electron to another, the protein complex is reduced and also  loses a proton.

There is a tendency to gain electrons 

H gradient  

- High concentration of protons found in the intermembrane space causes the electrochemical proton  gradient 

o H+ gradient is caused by proton pumping across the inner membrane space.

- Protons move from intermembrane space to the matric 

29

o Move from lower pH to higher pH 

o Move from higher concentration of protons in the intermembrane space to an area of lower  concentration of protons in the matrix

o Voltage gradient: High positive charge in intermembrane space, high negative charge in the  matrix 

- These forces will favour the movement of electrons from intermembrane space to the matrix  - Protons cannot go through inner membrane to matrix without facilitated transport because they have a  charge and ions need a transporter/ channel 

- High electrochemical gradient creates energy necessary for ATP synthase to use it to make ATP - Result

o Two forces that favour the movement of protons from intermembrane space to matrix through  ATP synthase 

o High electrochemical proton gradient caused by 

 Favourable movement of protons from high to low concentration 

 Favourable movement of electrons from high positively charged area to high negatively  charged area 

Electrochemical proton gradient  

- ATP synthase is found in the inner­membrane of the mitochondria 

o Large unit of multiple proteins 

o Regulates the process of phosphorylation of ADP  ATP

- It uses the energy from the high electrochemical gradient to produce ATP from ADP  - Proton passage through carrier (ATP synthase) causes the rotation of the central stalk  o Protons pass through the carriers rotator ring 

o Causing rotation of central stalk 

- This rotation alter the protein conformation and results in ATP production 

o The conformation change of the F1 ATPase head causes the addition of a phosphate group to  ADP  ATP

- If process was unregulated 

o Protons would be able to go through the channels without help and they could just flow back into the intermembrane space. 

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o This would mean there wouldn’t be any use of energy created by the electrochemical gradient  and thus there would be no production of ATP 

- So the flow of protons through ATPase allows ATPase to use the energy and produce 100 ATP  molecules/ second 

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Chapter 15 – intracellular compartments and protein transport  

Key points  

LDL uptake, protein transport via vesicle, Disulfide bond formation, Unfolded protein response, Vesicle  Docking, location of SRP, RAN nuclear transport, Signal peptidase, translocators and cytosolic fibrils, Golgi  structure, single pass and double pass protein diagram and vesicle transport, 

LDL uptake  

- Cholesterol is a lipid that is extremely insoluble in water. It is transported in the blood  stream bound to protein in the form of particles called low-density lipoproteins (LDLs) - Cholesterol containing LDLs which are secreted by the liver, bind to receptors located on  the cell surfaces causing the receptors located on cell surfaces causing the receptor LDL  complexes to be ingested by receptor mediated endocytosis and delivered to endosomes.  - The interior of endosomes is more acidic than the cytosol, here LDL dissociates from its  receptor:  

o the receptors are returned in transport vesicles to the plasma membrane for reuse  o the LDL is delivered to lysosomes  

- in the lysosomes, the LDL is broken down by hydrolytic enzymes  

- the cholesterol is released and escapes into the cytosol where it is available for new  membrane synthesis.  

Pathway for cholesterol/ LDL uptake  

- LDL entersn cells via receptor mediated endocytosis  

- LDL binds to LDL receptors on the cell surface and is internalized in clathrin coated  vesicles  

- The vesicles lose their coat and then fuse with endosomes  

- In the acidic environment of the endosome, LDL dissociates from its receptors  - The LDL ends up in lysosomes, where it is degraded to release free cholesterol, but the  LDL receptors are returned to the plasma membrane via transport vesicles to be used  again.  

Disulfide bond formation  

- Don’t alter protein structure but they do stabilize the protein  

- They are formed between sulfurs, you need a cysteine side chain because they only form  between them  

- Formation requires oxidizing environment, thus they are usually only formed in the ER  - The cytosol has a reducing environment and so these disulfide bonds cannot form in the  cytosol

o The cytosol has a reducing environment and so these disulfide bonds cannot form in the cytosol

- In the ER lumen, protein fold up, assemble with their protein partners, form disulfide  bonds, and become decorated with oligosaccharide chains

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-

Protein translocation to the ER

1. A mitochondrion has an outer and inner membrane, both of which must be crossed for a  mitochondrial precursor protein to enter the organelle  

2. To initiate transport, the mitochondrial signal sequence on a mitochondrial precursor  protein is recognized by a receptor in the outer mitochondrial membrane  3. This receptor is associated with a protein translocator  

4. The complex of receptor, precursor protein and translocator diffuse laterally in the outer  membrane until it encounters a second translocator in the inner membrane  5. The two translocators then transport the protein across both the outer and inner  membranes unfolding the protein in the process  

a. Transport to a particular site within the organelle requires further sorting signals on  the membrane.  

b. These signals are often only exposed after the first signal sequence has been  removed.  

6. The signal sequence is finally cleaved off by a signal peptidase enzyme in the  mitochondrial matrix.  

a. Proteins are imported into chloroplasts by a similar mechanism  

7. The chaperone proteins that help pull the protein across the membranes, into the matrix,  help the protein to refold  

Signal sequences  

- Amino acid sequences direct proteins to their destination 

- Once proteins are made, they require a signal sequence to be move to their final  destination.

- Signal sequences are recognized by specific receptors.

- These receptors are each destined to go to a specific site in the cell.

- The signals are similar to addresses on a shipping label.

- If the protein is tagged to go to a specific location the receptor will recognize the address  and take it there, regardless of the proteins actual composition.  

- Signals can also be used to take a soluble protein and bind it to its membrane. 

- It is possible to take a signal from ER and put it on a protein through the use of recombination and  mutations which would allow you to cause a change in the signal sequence. But it has to happen at the  DNA level 

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- It is much easier to manipulate at DNA level and at protein or RNA level.  - By using genetic engineering, you can change the gene which codes for an amino acid  sequence and therefore the change will appear in the protein.

- This is a permanent change.

Mitochondrial protein transport

- The proteins destined for mitochondria are pre­cursor proteins 

1. A protein is synthesized in the cytosol

2. A receptor protein on the mitochondrial membrane recognized the signal sequence on the  polypeptide chain and binds to it.  

3. The protein is linearized as it moves through the mitochondrial membrane.  4. Once inside the signal is cleaved off  

5. Chaperone proteins in the mitochondrial matrix help fold the protein into its final  conformation.  

- ER serves as an entry point for proteins destined for other organnels and the ER as well  - Proteins destined for the Golgi, endosomes, lysosomes and the cell surface all enter the ER from the cytosol first  

- Once inside the ER Lumen, ot embedded in the ER membrane, individual proteins will no  re-enter the cytosol they will be transported to their destinations via vesicles  - Two kinds of proteins are transferred from the cytosol into the ER  

o Water-soluble proteins are completely translocated across the ER  membrane and are released into the ER lumen  

 Destined for secretion – by release at the cell surface  

 Destined for the lumen of an organelle of the endomembrane system o Prospective transmembrane proteins are only partly translocated across  the ER membrane and become embedded in it.  

 Are destined to reside in the membrane of one of these organelles ot in the plasma membrane.  

- All of these proteins are initially directed to the ER by an ER signal sequence o 8 or more hydrophobic amino acids  

o Involved in the process of translocation across the membrane  

- Most proteins that enter the ER begin to be threaded through before the polypeptide chain has been completely synthesized.  

- This requires that the ribosome synthesizing the protein be attached to the ER membrane  - These membrane bound ribosomes coat the surface of the ER, creating regions termed  rough endoplasmic reticulum.  

Peroxisome

- Are involved in the breakdown of fatty acids, alcohol and toxins.  

- Their signal sequences are 3 amino acids long and there is no conformational change  needed to the protein to be translocated into peroxisome.

- They have a translocator protein which translocate itself with the protein into the  peroxisome.

- The peroxisome can also accept proteins through transport vesicles from the ER. Which  fuse with existing peroxisome  

o Or the vesicles will convert into mature peroxisome

- Peroxisomes acquire most of their proteins via selective transport from the cytosol. - A short sequence of 3 amino acids serves as an import signal for peroxisomes. The signal  sequence is recognized by receptor proteins in the cytosol. The peroxisome membrane  contains a translocator which helps transport into the cell  

o Proteins do not unfold to enter the peroxisome.

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Transport vesicles

- Budding transport vesicles have distinct coat proteins on their cytosolic surface; the  assembly of the coat helps drive both the budding process and the incorporation of cargo  receptors, with their bound cargo molecules, into the forming vesicle

- Vesicles that bud from membranes usually have a distinctive protein coat on their  cytosolic surface and are called coated vesicles  

- A few membrane proteins for peroxisome arrive via vesicles that bud from the ER  membrane  

- The vesicles either fuse with pre-existing peroxisomes or import peroxisome proteins from  the cytosol to grow into mature proteins.  

- For transport between organelles within the endomembrane system or to the plasma  membrane  

Unfolded protein response  

- Chaperons help proteins fold properly and retains those that do not in the ER  - When the control system becomes overwhelmed, misfolded proteins accumulate in the ER. If the buildup is large enough, it triggers a complex program called the unfolded protein  response  

- This program prompts the cell to produce more ER, including more chaperons and other  proteins concerned with quality control  

- The UPR allows a cell to adjust the size of its ER according to the load of the proteins  entering the secretory pathway.  

- In some cases, an expanded ER cannot keep up and the UPR directs the cell to self destruct by undergoing apoptosis.  

Secretory proteins are released from the cell by exocytosis  - A stream of vesicles bud from the trans Golgi network and fuses with the plasma  membrane in the process of exocytosis.

o This constitutive exocytosis pathway supplies the plasma membrane with newly made lipids and proteins, enabling the plasma membrane to expand prior to cell  division and refreshing old lipids and proteins in nonproliferating cells  

- The constitutive pathway also carries soluble proteins to the cell surface to be released to  the outside, a process called secretion .

o Some of these proteins remain attached to the cell surface, some are incorporated  into the extracellular matrix; others diffuse into the extracellular fluid to nourish or  signal other cells.

- Entry into the constitutive pathway does not require a signal sequence.  

Regulated exocytosis pathway  

- Operated only in cells specialized for secretion.  

- Each specialized cell produces large quantities of a particular product which is sorted in  secretory vesicles for later release.

- These vesicles which are part of the endomembrane system bud off from the trans Golgi  network and accumulate a signal that will stimulate them to fuse with the plasma  membrane and release their contents to the cell exterior by exocytosis.  

- Proteins destined for regulated secretion are stored and packaged in the trans Golgi  network. Proteins that travel by this pathway have surface properties that allow them to  aggregate with one another under the ionic conditions (acidic pH and high Ca2+) that  prevail in the trans Golgi network.

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Golgi structure  

- recieves newly made proteins from the ER; it modifies their oligosaccharides, sorts the  proteins, and dispatches them from the trans Golgi network to the plasma membrane,  lysosomes (via endosomes), or secretory vesicles.  

- The Golgi is located near the cell nucleus  

- Consists of a collection of flattened, membrane enclosed sacs called cisternae, which each contain 3-20 cisternae  

- The number of sacs per cell varies depending on the cell type  

- Each Golgi each has two faces  

o A cis-face/ an entry  

o And a trans-face/ an exit  

- The cis-face is adjacent to the ER, while the trans face points toward the plasma  membrane.  

- The outermost cisterna at each face is connected to a network of interconnected  membranous tubes and vesicles.  

- Soluble proteins and membrane enter the cis Golgi network via transport vesicles derived  from the ER

- The proteins travel through the cisternae in sequence by means of transport vesicles that  bud from one cisterna and fuse with the next.  

- Proteins exit from the trans Golgi network in transport vesicles destined for either the cell  surface or another organelle of the endomembrane system  

- Both the cis and trans Golgi networks are important for protein sorting: o Proteins entering the cis Golgi network can either move onward through the Golgi  stack or,  

o if they contain an ER retention signal, can be returned to the ER o proteins exiting from the trans Golgi network are sorted according to whether they  are destined for lysosomes (via endosomes) or for the cell surface.  

- Many of the oligosaccharide chains that are added to proteins in the ER undergo further  modifications in the Golgi.  

- On some proteins, more complex oligosaccharide chains are created by a highly ordered  process in which sugars are added and removed by a series of enzymes that act in a  rigidly determined sequence as the protein passes through the Golgi stack.  

- The aggregated proteins are packaged into secretory vesicles with which pinch off from  the network and await signal instructing them to fuse with the plasma membrane  - These proteins have selective aggregation which allows secretory proteins to be packaged  into secretory vesicles at concentrations much higher than the concentration of  unaggregated protein in the Golgi lumen.  

Arrangement of a transmembrane protein in the lipid bilayer  - A soluble protein crosses the ER membrane and enters the lumen  

- The protein translocator binds the signal sequence and threads the rest of the polypeptide  across the lipid bilayer as a loop.  

- At some point during the translocation process, the signal peptide is cleaved from the  growing protein by a signal peptidase.  

- This cleaved signal sequence is ejected into the bilayer where it is degraded.  - Once protein synthesis is complete, the translocated polypeptide is released as a soluble  protein into the ER lumen, and the pore of the translocation channel closes.  

single membrane pass

- The N-terminal signal sequence initiates translocation. But the transfer process is halted  by an additional signal sequence of hydrophobic amino acids, a stop-transfer sequence firther along the chain

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- The translocation channel then releases the growing polypeptide chain into the lipid  bilayer.  

- The N-terminal signal sequence is cleaved off, the stop-transfer sequene remains in the  bilayer where is forms an alpha-helical membrane spanning segment that  anchors the protein in the membrane  

- The protein ends up as a single-pass transmembrane protein interted in the membrane  with a defined orientation  

o The N-terminus on the luminal side of the lipid bilayer and the C-terminus on the  cytosolic side.  

1. A single pass membrane protein is retained in the lipid bilayer.  

2. An N-terminal ER signal sequence initiates transfer.  

3. The protein also contains a second hydrophobic sequence which acts as a stop-transfer  sequence  

4. When this sequence enters the translocation channel, the channel discharges the growing  polypeptide chain sideways into the lipid bilayer.  

5. The N-terminal signal sequence is cleaved off, leaving the transmembrane protein  anchored in the membrane.  

6. Protein synthesis on the cytosolic side then continues to completion.  

double membrane pass

- In some transmembrane proteins the internal signal sequence is used to start the  protein transfer this signal sequence is called the start-transfer sequence and is  never removed from the growing chain  

- This causes the polypeptide chain to pass back and forth across the lipid bilayer. Here the  hydrophobic signal sequences work in pairs.  

o An internal start-transfer sequence serves to initiate translocation which continues  until a stop-transfer sequence is reached.  

o The two hydrophobic sequences are then released into the bilayer, where they  remain as membrane spanning alpha helices.  

- A double pass transmembrane protein has an internal ER signal sequence  o This sequence acts as a start-transfer signal and helps to anchor the final protein in  the membrane  

o The internal signal sequence is recognized by an SRP, which brings the ribosome to  the ER membrane  

o When a stop-transfer sequence enters the translocation channel, the channel  discharged both sequences into the lipid bilayer.

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o Neither the star-transfer nor the stop-transfer sequence is cleaved off and the entire chain remains anchored in the membrane as a double-pass transmembrane  - Proteins that span the membrane more times contain further pairs of start and  stop transfer sequences, and the same process is repeated for each pair. Localization signals and recognition particles  

RECEPTOR RECEPTOR LOCATION

SIGNAL 

DESTINATION

SIGNAL 

STRUCTURE

FUNCTION  SIGNAL

NUCLEAR IMPORT RECEPTOR

Cytosol Nucleus  Not done yet  Binds to nuclear protein with  localization signal. Transports into the 

nucleus and releases the protein 

Not 

cleaved 

RAN­GTP In nucleus  Binds to nuclear import receptor to  translocate it out of the nucleus 

Binding to receptor causes conformation 

change. It is then hydrolyzed to cause 

another conformation change to release 

receptor

MITOCHONDRIA  TRANSLOCATOR  PROTEINS 

cytosol Mitochondria  Protein is  linearized

Recognizes signal sequence on protein  (conformation change) binds to protein.  Translocated and binds to outer  mitochondrial membrane (conformation  change) membrane passes the protein to  translocators on outer and inner  mitochondrial membrane 

Cleaved 

LIPID CARRYING  Cytosol Peroxisomes  A sequence of  Sequence signal recognized by receptor  Don’t 

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PROTEINS  3 amino acids 

proteins in cytosol. They bind the signal 

cleave

SRP RECEPTOR   Cytosol Endoplasmic  reticulum

signals 

transport to  peroxisomes  ER signal 

sequence is 8  or more amino  acids long 

and move it in through protein  translocators into peroxisome

No conformation change 

Translocation of proteins takes place as a protein is being translated. (occur co translationally) 

Free ribosomes bind to signal sequence,  translocate molecule to ER where they  bind to the membrane 

cleaved

- Most organelle proteins are made in the cytosol and transported into the  organelle where they function. Sorting signals in the amino acid sequence guide the proteins to the correct organelle; proteins that function in the cytosol have  no such signals and remain where they are made.

- Nuclear proteins contain nuclear localization signals that help direct their active transport  from the cytosol into the nucleus through nuclear pores, which penetrate the double membrane nuclear envelope. The proteins are transported in their fully folded  conformation  

- Most mitochondrial and chloroplast proteins are made in the cytosol and are then  transported into the organelles by protein translocators in their membranes. The proteins  are unfolded during the transport process.  

- The ER makes most of the cells lipids and many of its proteins. The proteins are made by  ribosomes that are directed to the ER by a signal-recognition particle SRP in the cytosol  that recognizes an ER signal sequence on the growing polypeptide chain. The ribosome SRP complex binds to a receptor on the ER membrane, which passes the ribosome to a  protein translocator that threads the growing polypeptide across the ER membrane  through a translocation channel.  

- Water soluble proteins destined for secretion or for the lumen of an organelle of the  endomembrane system pass completely into the ER lumen, while transmembrane proteins destined for either the membrane of these organelles or for the plasma membrane remain  anchored in the lipid bilayer by one or more membrane spanning alpha helices  

Nuclear pores  

- Composed of 30 proteins  

- Mostly unstructured regions which extend out of the nucleus  

- Responsible for movement of protein and nuclear import receptor  

Nuclear protein transport

Nuclear Pore import

1. Nuclear protein has localization signal  

2. The signal is recognized by nuclear import receptor in cytosol  

3. The nuclear import receptor binds to the nuclear protein which forms a complex  4. The complex will interact with cytosolic fibers. The interactions weaken the fibers and  there is a conformation change

a. If there is no signal and the complex doesn’t bind, the cytosolic fibers will interact  with each other to keep the structure closed and will not allow passage into the  nucleus.

b. So in order to pass into the nucleus, the complex binds and causes a conformation  change

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5. When weakened, the complex can go through the nuclear basket because the nuclear pore opens  

a. So conformation change causes nuclear pore to open and the complex can move  through the nuclear basket into the nucleus  

6. Nuclear receptor complex interacts with the nuclear basket, this causes the nuclear import receptor protein to change its conformation so it releases the protein bound to it. 7. The signal remains intact on the internis once the protein is released into the nucleus. a. Nuclear transport signal is not cleaved.

b. Protein is recycled by returning to cytosol.  

Nuclear Pore export and RAN-GTP

- Movement of receptor out of the nucleus is through binding to RAN­GTP

- Binding to the receptor provides energy for protein import into nucleus  - In order for the receptor to exit the nucleus it must be bound to RAN-GTP 1. In nucleus the receptor releases the protein and binds to RAN-GTP

2. The RAN-GTP moves out of the nucleus  

3. Once out of the nucleus, the RAN-GTP is hydrolyzed  

4. This hydrolysis causes conformation change of RAN-GTP so it releases the Nuclear import  receptor  

5. This hydrolysis also provides energy for RAN-GTP to translocate back into the nucleus  - Export is important because if the protein was allowed to accumulate in the nucleus rather than returning  to the cytosol it would cause problems for the cell.

SRP – in the cytosol

- Two protein components help guide ER signal sequence to the ER membrane  1. A signal recognition particle (SRP) in the cytosol  

a. binds to both the ribosome and the ER signal sequence when it emerges from the  ribosome  

2. an SRP receptor  

a. embedded in the ER membrane  

b. recognizes SRP

c. binding of an SRP to a ribosome that displays an ER signal sequence slows protein  synthesis by that ribosome until the SRP engages with an SRP receptor on the ER d. once bound, the SRP is released  

e. the receptor passes the ribosome to a protein translocator in the ER membrane  f. protein synthesis recommences  

g. the polypeptide is then threaded across the ER membrane through a channel in the  translocator  

- The translation process is slowed down. During translocation  

o The movement happens at the same time as translation = co

translationally

- The ribosome then bonds to the ER membrane and the process of translation continues at  normal speed on a ribosome attached to the ER membrane  

- The whole complex is then passed onto the SRP receptor which recognized the recognition particle and displaces itself with the complex to then release the complex onto a protein  translocator  

- Once ribosome is on the protein translocator transcription resumes at normal speed  allowing the growing polypeptide to be synthesized into the ER lumen  

- The signal sequence also functions to open channels in the protein translocator  o This sequence remains bound to the channel, while the rest of the polypeptide chain is threaded through the membrane as a large loop.

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o It is removed by a transmembrane signal peptidase, which has an active site facing  the luminal side of the ER membrane  

o The cleaved signal sequence is then released from the translocation channel into  the lipid bilayer and rapidly degraded  

- Once the C-Terminus of a soluble protein has passed through the translocation channel,  the protein will be released into the ER lumen  

Clathrin coated proteins

- Coated vesicles rapidly lose their protein coat, enabling them to dock and then fuse with a  particular target membrane; docking and fusion are mediated by proteins on the surface  of the vesicle and target membrane, including Rab and SNARE proteins.  

- Bud from both the Golgi on the outward endocyclic pathway and the plasma membrane on the inward endocytic pathway

- At the plasma membrane  

o Each vesicle starts as a Clathrin coated pit. The clathrin molecules assemble into a  network on the cytosolic surface of the membrane.  

o A small GTP-binding protein called dynamin assembled as a ring around the neck of each deeply coated pit. Together with other proteins recruited to the neck of the  vesicle, the dynamin causes the ring to constrict, thereby pinching off the vesicle  from its parent membrane.  

- Clathrin does not choose the specific molecules for transport. This is the function of  Adaptins

o Adaptins secure the clathrin coat to the vesicle membrane and help select cargo  molecules for transport.  

o Molecules for onward transport carry specific transport signals that are recognized  by cargo receptors in the Golgi and plasma membrane.  

o Adaptins help capture specific cargo molecules by trapping the cargo receptors that  bind them so that a selected set of cargo molecule, bound to specific receptors is  incorporated into the lumen of each newly formed clathrin coated vesicle.  

- Once the vesicle has reached its target it must recognize and dock with its specific  organelle.  

o Then the vesicle membrane fuses with the target membrane and unloads the cargo  molecules.  

GTPases

- Rab proteins are specific to each type of vesicle.  

- Specific Rab proteins are recognized by corresponding tethering proteins on the cytosolic  surface of the target membrane.  

- Each organelle and each type of transport vesicle carries a unique combination of Rab  proteins, which serve as molecular markers for each membrane type.  

o This ensures that transport vesicles only fuse with the correct membrane type  

SNAREs  

- Additional recognition is provided by SNAREs  

- Once the tethering protein has captured a vesicle by grabbing hold of its Rab protein,  SNAREs on the vesicle interact with complementary t-SNAREs on the target membrane.  

Fusion

- The same SNAREs involved in docking also play a role in catalyzing the membrane fusion  requires for a transport vesicle to deliver its cargo.  

- Fusion also adds the vesicle membrane to the membrane of the organelle.

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- After vesicle docking the fusion of a vesicle with its target molecule requires a stimulatory  signal.  

- Requires the lipids of the two bilayers to intermix. And water must be displaced from the  hydrophilic surfaces of the membrane.  

o This process is highly energetically favourable and prevents membrane from fusing  randomly  

- All membrane fusions must be catalyzed by proteins that form a fusion complex which  provides the means to cross the energy barrier.

- The SNARE proteins catalyze the fusion process  

o Once fusion is triggered, the v-SNAREs and the t-SNAREs wrap around each other,  and pulls the two lipid bilayers into close proximity  

Transcytosis

- The endosomal compartment acts as the main sorting station in the inward endocytic  pathway, just as the trans Golgi network serves this function in the outward secretory  pathway. The acidic environment of the endosome plays a crucial part in the sorting  process by causing many (but not all) receptors to release their bound cargo. The routes  taken by receptors once they have entered an endosome differ according to the type of  receptor:  

o (1) most are returned to the same plasma membrane domain from which they  came, as is the case for the LDL receptor dis- cussed earlier;  

o (2) some travel to lysosomes, where they are degraded; and  

o (3) some proceed to a different domain of the plasma membrane, thereby  transferring their bound cargo molecules across the cell from one extracellular  space to another, a process called transcytosis

- The fate of receptor proteins following their endocytosis depends on the type of receptor. - receptors that are not specifically retrieved from early endosomes follow the pathway  from the endosomal compartment to lysosomes, where they are degraded.  - retrieved receptors are returned either to the same plasma membrane domain from which  they came (recycling) or to a different domain of the plasma membrane (transcytosis).  - tight junctions separate the apical and basolateral plasma membranes preventing their  resident receptor proteins from diffusing from one domain to another. If the ligand that is  endocytosed with its receptor stays bound to the receptor in the acidic environment of the  endosome, it will follow the same pathway as the receptor; otherwise it will be delivered to lysosomes for degradation.

- The nuclear pore complex forms a gate through which selected macromolecules  and larger complexes enter or exit the nucleus  

o Protein fibrils protrude from both sides of the pore complex in the nuclear envelope.   On the nuclear side, they converge to form a basketlike structure  

 The spacing between the fibrils is wide enough that the fibrils do not obstruct  access to the pore.  

o The nuclear loc

- The outer membrane is continuous with the ER membrane  

o The double membrane of the nuclear envelope is penetrated by nuclear pores.  - The macromolecule complexes must display an appropriate sorting signal. The signal  sequence that directs a protein from the cytosol into the nucleus, called a nuclear  localization signal, typically consists of one or two short sequences containing several  positively charged lysine or arginine’s  

- The nuclear localization signal on proteins destined for the nucleus is recognized by  cytosolic proteins called nuclear import receptors  

o These receptors help direct a newly synthesized protein to a nuclear pore by  interacting with the cytosolic fibrils that extend from the rim of the pore into the  cytosol.

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o Once there the nuclear import receptor penetrated the pore by grabbing onto short  repeated amino acid sequences within the tangle of nuclear pore proteins that fill  the center of the pore.  

At the translocator

- Soluble proteins are released into the ER lumen  

- The growing polypeptide chain will go through translocator which causes the signal  sequence (os a soluble protein) to be cleaved  

- The signal peptidase enzyme in the ER membrane with its active site in the ER lumen will  cleave the signal sequence

- Causing the whole translation process to complete and the mature soluble protein will be  released into the ER lumen  

- The cleaved signal peptide will be recycled back into the cytosol.  

-

Signal peptidase enzyme

- The signal peptidase enzyme is responsible for removing and cleaving the signal sequence found on the polypeptide chain of a soluble protein destined for the ER lumen.  - The signal peptidase enzyme is bound to the ER outer membrane. Once the peptide bind  to its active site the enzyme will cleave off the signal sequence to the peptide can be  translocated into the ER lumen.

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Ch. 18 – the cell cycle  

Key points  

Mitogen, APC targets, securing, separase, cell cycle checkpoints for replication and DNA damage, centrosome  duplication, cohesin, cytokinesis, organelle division during cell cycle, contractile ring, myosin, actin,  microtubules, apoptosis (Bax, Bak, Cytochrome c, Bcl2, apoptosome), cyclin and Cdk role in cell cycle,  centrosome cycle, Wee1, Cdc25, different microtubules involved in Metaphase and Anaphase, kinetochore,  spindle, 

The cell cycle – overview  

- Is made up of phases. The purpose is to duplicate DNA and segregate genetic information  identically into two daughter cells from one parent cell.  

- The time is takes to do the cycle varies between animals and organisms, and cell sizes and types  

o For e-coli it takes 30 minutes  

o Yeast cells 1.5 hours  

o Mammalian intestinal epithelial cells 12 hours  

o Mammalian fibroblasts in culture 20 hours  

Cell cycle phases

- M-phase  

o The shorter phase  

o Made up of  

 Mitosis – when the nucleus divides

 Cytokinesis – when the cell splits in two  

- Interphase

o The longer phase – between one M-phase and the next  

o Made up of  

 G1

 G2

 S

Cell cycle control system

- The system guarantees that the events in the cell cycle occur in a set sequence and that  each process has completed correctly before the next one can begin.

o To regulate this, certain critical points of the cycle feedback from the process  currently being performed.  

o DNA damage is checked for at every checkpoint

o Check for errors that may have occurred during the previous step before moving  onto the next step.  

- Checkpoints pause the cell cycle and checks for errors and sources of errors  o It will then fix any errors it finds  

- Checkpoints  

o Transition from G1 to S

 Confirms the environment is favorable for proliferation before committing to  

DNA replication.

o S to G2

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 Checks to see if environment is favorable.  

 If not it will delay progress through G1 and can enter G0

o G2 to M  

 Is all DNA replicated  

 Is all DNA damage repaired  

o Mitosis

 Duplicated chromosomes are properly attached to a cytoskeletal machine  (mitotic spindle)

Cyclin-cdk’s

- The cell-cycle control system depends on cyclically activated protein kinases called Cdks  (cyclin dependant protein kinases)

o Carried out largely through phosphorylation and dephosphorylation

- Kinases and phosphatases control and regulate cell cycle control proteins.  o In order to activate the kinases and phosphatases, cyclin needs to be present.  o A Cdk must bind to a protein called cyclin before it can become enzymatically  active. This activation also requires an activating phosphorylation of Cdk

- Cyclin concentration fluctuates throughout the entire cycle to control and regulate the  activity of the cell cycle. The cyclical changes in concentration help drive the activation of  the compelxes.  

1. M-cdk (cyclin dependant kinases) activates cyclin by physically binding to it.  Once M-cdk binds to cyclin it forms a complex  

2. Cyclin concentration increases when CDK concentration increases.  - If cyclin concentration was always high, the cell would continue to cycle through the cell  cycle and continue to go through the phases without stopping at the checkpoints.  o When there is no cyclin, there is no activity of M-CDK

o No MCDK activity = the cell will not commit to the next phase.  

Example: if the cell is not ready to commit to s­phase one of the following should occur.

o S-Cyclin levels  

o CDK activity  

o Combination of both the above by regulating the complex.

There are different combinations of CDK­cyclin complexes that form to control specific events that take place at different  phases. 

- Formation of different complexes occurs through the binding of different CDK’s to different  cyclins.  

- Example: S-Phase is S-CDK dependant.

o S-CDK (consists of CDK and cyclin A) causes DNA synthesis  

o If SCDK is not present then the DNA is not synthesized.  

o This occurs when the environment is not ready, there is no intercellular signaling, or in the case of unicellular organisms, when there is not enough nutrition.

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- Example question: if there is incomplete replication, what does the cell  regulate? S-CDK, G1-CDK, or M-CDK?

o The cell would regulate M-CDK. Because if DNA has already been synthesized, then  S-CDK, and G1,CDK has already been activated.  

o Therefore; M-CDK regulation will prevent DNA from entering and committing to M Phase.  

Cyclin regulation  

- Protein concentration is determined by the rate of protein synthesis over the rate of  protein degradation.  

- APC regulates cyclin degradation.

o APC is active during the M phase. It regulates cyclin by degradation if spindle  formation is not complete.  

- Cyclin degradation drives transition from one phase to the next.  

- The cell can control cyclin levels by  

1. Degrade proteins  

2. Controlling the concentration of proteins  

3. Inhibiting gene synthesis of the protein  

4. Transcription factors (phosphorylation/methylation)

5. Inhibiting cell communication.  

- Example: degradation of Cyclin  

o If cdk or cyclin is degraded then there is no activity.  

o The NFA promoting complex (APC) regulates cyclin by degradation  

o Degradation of cyclin regulates S-CDK and M-CDK activity  

1. APC targets and tags cyclin with a ubiquitin chain

2. APC signals for degradation by calling ubiquitin chain in.  

3. causes ubiquitylation (adds ubiquitin chain) of Cyclin proteasome

a. Ubiquitylation causes inactivation of cyclin  

4. Degradation of cyclin causes Cdk to remain inactive because there is no cyclin present to  activate it.  

Cdk-Cyclin activity  

- If DNA replication is not properly completed, Wee1 phosphorylated cdk  - A form of protein modification to regulate checkpoints  

- Inhibitory phosphates deactivate Cdk-cyclin (phosphorylation of cdk-cyclin = inactivation) - Example: Phosphorylation of M-CDK

1. DNA is not synthesized properly during S-Phase

2. Wee1 phosphorylates mitotic Cdk  

3. Mitotic Cdk binds to M cyclin = M-cdk complex

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a. Binding of phosphorylated M-CDK to M-cyclin inactives M-cyclin  

4. With inactivated M-CDK complex the cell cannot commit to M-phase  

- Example: activation of cyclin-Cdk complex – Occurs in G1-CDKs and S-CDKs o P27 is a phosphatase protein (removes phosphates)

 P27 is present during G1-Phase and S-Phase. Will stop cycle is environment is not ready or if cell growth is not sufficient (won’t dephosphorylate the  

complex)

o P27 binds to the complex (forms a physical interaction) and removes inhibitory  phosphates from M-CDK  

o Dephosphorylation causes activation of CDK complex  

o Active M-CDK allows cell to enter and commit to the M-Phase  

Figure: P27 binds to cyclin­cdk complex to dephosphorylate and activate the complex at S­ and G1­Phase checkpoints 

G1 Phase  

- Cell growth, metabolic activity and repair.

o Cell prepares to be duplicated during G1 phase by  

 Enhancing formation of proteins involved

 Growing ER and Golgi  

 Enhancing ATP synthesis by increasing activity of mitochondria.

- Cells at G1 enter S phase, remain in G1, or enter G0 depending on cell size or extracellular  signaling.  

o In unicellular organisms  

 Availability of nutrition regulates the phase the cell will enter  

o In multicellular organisms  

 Environment and other factors (listed above) determine the phase the cell will

enter  

- M and S cyclin CDK complexes must be inactivated before entry into G1 phase. (result is  inactive cyclin at G1-Phase)

o Regulation occurs through degradation (elimination) of cyclin proteins  o Inhibition of protein synthesis (blocks the synthesis of new S-CDK and M-CDK) o Or inhibitory proteins (Wee1) target CDK or Cyclin  

o Or muffles the activity of any remaining Cyclin-cdk complexes

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Mitogens (a form of extracellular signals)

- Once the cell receives mitogen signal, transcription regulators initiate  transcription of various genes involved in cell proliferation  

- Promote the production of the cyclins that simulate cell division

o If deprived of these signals, the cell cycles arrests in G1

o If deprived of these signals for an extended period of time, the cell will withdraw  from the cell cycle and enter a nonproliferating state.

- To escape from cell arrest  

o To escapes from cell arrest requires accumulation of cyclins.

o Mitogens act by switching on cell signaling pathways that stimulate the synthesis of  G1 cyclins and other proteins involved in DNA synthesis and chromosome  duplication.

o The build up of cyclins triggers a wave of G1/S-Cdk activity, which relieves the  negative controls that are preventing entry to S-phase

- Stimulate multiplication of mammalian cells

o Cause: Trigger synthesis of G1 and G1/S-cyclin  

 Effect: increase cyclin concentration in cell = more cyclin bind to more CDK=  

more active CDK-cyclin complexes  

o Cause: activation of G1 cyclins  

 Effect: increased synthesis of proteins involved in DNA replication  

- Inhibition of Mitogens occurs through Rb proteins  

o Cause: Absence of extracellular signals like mitogens  

 Effect: Rb (retinoblastoma) binds to transcription regulators to inactivate  

them  

 Effect: prevents synthesis of G1 and G1/S-cyclin and other proteins involved  

in replication  

- Explanation: Mitogen absent

o No extracellular signals= Rb binds to transcription regulators  

o = no protein synthesis = no protein expression = no cycle, the cell arrests in G1- Phase

- Mitogen present- escape from cell arrest in G1

o Cell receives signal by mitogen binding to receptors on cell membrane  o Intracellular signaling through cell activates G1-cdks (a kinase protein) o = G1-CDK phosphorylates Rb to inactivate it = transcription regulators are released o = transcription regulators are active and bind to DNA to start transcription of genes  for proteins involved in the cell cycle. = metabolic activity enhanced

o = Cell commits to S-phase

- A mutation to the Rb protein causes childhood eye tumors  

DNA damage  

- p53 function in the DNA damage response is to increase the expression of p21  - If DNA is damaged, the cell cycle progression is paused (checkpoint detects damage) - At G1-to-S transition (prevents the cell from replicating damaged DNA)  

o DNA damage in G1-Phase causes an increase in concentration and activity of P53  - P53 fixed DNA damage during the G1 phase

- Induction of DNA damage in G1 to S-phase  

o Kinases that phosphorylate P53 are activated by DNA damage detection.  o P53 (transcription regulator for P21) is activated by phosphorylation  

o Activated P53 binds to regulatory region on the P21 gene to begin synthesis of P21  (Cdk inhibitor protein)

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o P21 is synthesized and binds to S-CDK and G1/S-CDK complexes to inactivate the  complex  

o Arrests the cell in G1=phase giving the cell time to repair the damaged DNA before  replicating it.  

- If DNA damage is too severe to repair, P53 can induce the cell to kill itself by undergoing  apoptosis  

- If DNA is not damaged, P53 is not activated, P21 is not synthesized, complex remains  active  

- When DNA damage is fixed, P21 will unbind from the complex so the cycle can continue to  the S-Phase  

Non-dividing state (G0 Phase)

- Cell division can stop for a prolonged-period  

o Nerve cells  

o Muscle cells

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- To enter G0 Phase there needs to be repression of genes encoding CDKS and cyclin.  o Shuts down production of CDK and cyclin to prevent cell from continuing through  

the cycle.  

- The G0 phase is temporary for some cells  

o Liver cells

- The Go phase can also be permanent wherein there is complete repression.  

S-phase  

- the centrosome cycle is the process of duplication and separation of  centrosomes which starts in S phase  

- cdc6 binds to ORC and recruits DNA helicase during the S phase  - S-Cdk initiates DNA replication and blocks re-replication – requires other factors o Origin of replication  

o Origin recognition complex (ORC)

 Recognizes and binds to the origin of replication  

 Recruits CDC6

o CDC6 (C6)

 Once ORC binds to the origin of replication (OR) C6 binds to the ORC=  

complex

 C6 helps in recruitment of helicase.  

 C6 and ORC together load the DNA helicase

 When helicase binds to complex, C6 is phosphorylated by S-CDK which  

causes C6 to dissociate  

o Helicase

 Opens the double helix to start synthesis.

o Pre-replication complex/ PREREPLICATIVE complex

 The complex is formed by ORC + helicase  

o S-CDK (cyclin-cdk complex that triggers S-Phase)

 Is assembled and activated at the end of G1-phase. Gives the initiation  

signal.

 Activates DNA helicase in the preplicative complex and promotes the  

assembly of the rest of the proteins that form the replication fork.  

 Triggers initiation of DNA synthesis by tagging C6 for degradation by  

phosphorylating C6  

 So when C6 is degraded, DNA synthesis begins  

 S-CDK phosphorylating C6 causes degradation is very important because this  is what prevents re-synthesis of the DNA.

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- Incomplete DNA synthesis can arrest the cell cycle in G2

o M-cdk is inhibited by phosphorylation at particular sites. Inhibitory phosphates must  be removed by an activating protein phosphatase called CDC25

o M-CDK phosphorylated so that It is not active so cell cannot enter the M-Phase - Cdc25 is inactive = cell arrests in cell cycle in G2

- When DNA is damaged or incompletely replicated, Cdc25 is inhibited, preventing the  removal of inhibitory phosphate.  

o As a result, M-Cdk is inhibited, preventing the removal of phosphates.  o M-Cdk remains inactive and M-Phase is delayed until DNA replication is complete - Cdc25 phosphotase activates M-Cdk

o Once a cell has replicated its DNA in S-phase, and progressed through G2, it is ready to enter M-Phase

o The cell will divide its nucleus (mitosis) and then its cytoplasm (cytokinesis).

M-Phase Overview

- Short relative to cell cycle  

o Consists of Mitosis and cytokinesis  

- During this period, the cell reorganizes all of its components and distributes them equally  into the two daughter cells.

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- The chromosomes must be accurately segregated so that each daughter cell has an  identical copy of the chromosome.  

- Cytoskeletal machines

o Microtubules Pull duplicated chromosomes apart (mitosis)

o Myosin and actin filaments = divide cytoplasm into two halves (cytokinesis)

Cdc25

- If DNA is damaged or incompletely replicated in the S phase, the inhibition of Cdc25  prevents the cell from entering M phase.  

- For M-Cdk to be activated, the Cdc25 phosphatase must remove the inhibitory phosphates from M-Cdk.

- When Cdc25 activates M-Cdk, a positive feedback loop is initiated, where M-Cdk  phosphorylates and further activates more Cdc25.  

- Therefore, keeping Cdc25 in an inactive state prevents the initiation of the positive  feedback loop and prevents cells from entering M phase  

- The Activation of M-Cdk begins abruptly because each M-Cdk complex can activate more  M-Cdk through a positive feedback mechanism. Activation of M-Cdk depends on the  removal of inhibitory phosphates by Cdc25. Activated M-cdk can phosphorylate and  therefore activate Cdc25, thus promoting activation of additional M-Cdk.  

Entry into M-Phase

- M-CDK regulates the early stage of M-Phase  

o By the assembly of the mitotic spindle  

- M-CDK is activated at the end of G2 by activating phosphatase Cdc25 which removes the  inhibitory phosphates holding M-Cdk activity in check.  

- M-cdk complex can indirectly activate additional M-Cdk complexes – by phosphorylating  and activating more cDC25.  

- Activated M-Cdk complex also shuts down the inhibitory kinase Wee1, further promoting  the production of activated M-Cdk.

- M-CDK phosphorylates CDC25 (C25) to activate it  

- C25 activated form blocks the inhibitory actions of kinase Wee1 to allow cell to enter and  commit to M-Phase  

o Wee1 inhibits cell from entering M-Phase (if there is incomplete DNA replication at  S-Phase) by phosphorylating M-CDK

- Creates a positive feedback loop where active M-CDK enhances production of itself  through phosphorylation of C25

- M-cdk is involved in the assembly of mitotic spindle and other proteins involved in the M Phase.

- M-CDK is accumulated during G2 but in an inactive form. Its activity requires C25  phosphatase.

- Overall: once M-Cdk activation begins, it causes an explosive increase in M-Cdk activity  that drives the cell abruptly from G2 to M phase.

- The process explained: Activation of M-CDK positive feedback loop

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1. Cdc25 (C25) phosphatase is activated by a protein (not M-CDK initially) 2. C25 removed phosphate on M-CDK = activation of M-CDK

3. Active M-CDK phosphorylated more C25 to activate = More C25

4. More C25 = more active M-CDK  

Cohesins and condensing (cytokinesis)

- Duplicated chromosomes in the nucleus are condensed at the M-Phase by condensins o Condensins are activated by MCDK activation

o Assemble along the length of each chromatid as the DNA is replicated. This  cohesion between sister chromatids is crucial for proper segregation, and it is  broken completely in the late mitosis to allow the sisters to be pulled apart by the  mitotic spindle.  

o Form ring around chromosomes to condense them in the nucleus.

- Cohesins hold sister chromatids(assembled in the S-Phase) together

o Very important because otherwise there could be an unequal amount of  chromosomes in two sister chromatids

o Form a ring around the sister chromatids to hold them together  

o Help condense the chromosomes which reduces mitotic chromosomes to compact  bodies that can be more easily segregated within the crowded confines of the  dividing cell.  

 The assembly of condensin complexes onto the DNA is triggered by the  

phosphorylation of condensins by M-Cdk.

- Cohesins and condensins are structurally related and both are thought to form ring  structures around chromosomal DNA. But cohesins assemble on each individual sister

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chromatid at the start of M-Phase and help each of these double helices to coil up into a  more compact form. Together they help configure replicated chromosomes to mitosis.

Cytoskeletal activity  

- During mitosis there is formation of mitotic spindle (carry out nuclear division) o Mitotic spindles are composed of microtubules and motor proteins

o Spindles stem from the organizing center

- During cytokinesis, there is formation of a contractile ring which carries out cytoplasmic  division  

o Actin and myosin filaments form the contractile ring in the cell cortex which creates  force around the cytosol and divides the cell into two sister cells.  

o As actin slides past myosin, it causes the plasma membrane to pinch, which  eventually results in the formation fo two separate cells.  

o Contractile ring consists mainly of actin filaments and myosin filaments arranged in  a ring around the equator of the cell. It starts to assemble just beneath the plasma  membrane toward the end of mitosis. As the ring contracts, it pulls inwards, thereby dividing a cell in two.  

-

Mitotic spindle (main event during prophase)

- The mitotic spindle begins to assemble during the initial stage of mitosis once interphase  has completed.

o Centrosomes (the organizing center where mitotic spindles originate from) are  duplicated at S-Phase.

o Duplication continues through M-Phase

- Microtubules continuously polymerize and depolymerize by the addition and loss of their  tubulin subunits and individual filaments alternate between growing and shrinking – a  process called dynamic instability.  

o At the start of mytosis, the stability decreases – in part because of M-CDK  phosphorylated mitotic associated proteins that influence the stability of the  microtubules

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 As a result, during prophase, rapidly growing and shrinking microtubules  extend in all directions from the two centrosomes, exploring the interior of  the cell.

- The assembly of the spindle is driven, in part, by motor proteins associated with the  interpolar microtubules that help to cross-link the two sets of microtubules.  - Spindle poles  

o Once the mitotic spindles are formed, the centrosome becomes the spindle pole. o Microtubules reach out to interact with and bind to microtubules on the other  centrosome. This interaction stabilizes the microtubules, preventing them from  depolymerizing and it joins the two sets of microtubules together to form the  basic framework of the mitotic spindle.

o The two centrosomes that give rise to these microtubules are now called  spindle pores.  

 And the interacting microtubules are called interpolar microtubules.  

- There are different types of microtubules  

o Interpolar microtubules  

 Attach to opposite spindle poles  

 Extend from one spindle pole to the opposite spindle pole  

 Form the interpolar microtubules (interacting microtubules)

o Aster microtubules  

 Extended away from the nucleus  

 Are not attached to anything.  

- ALTHOUGH there are different types, they are similar  

o All microtubules are extended  

o All attach to different parts  

o All together form the mitotic spindle.

Chromosome attachment (main event from prometaphase) - The key event in prometaphase is phosphorylation of the nuclear lamin protein  - Microtubules capture chromosomes by binding to kinetochores on the sister  chromatids  

- Disassembly of nuclear envelope (into small membrane vesicle) starts at prometaphase  o Process triggered by the phosphorylation and consequently disassembly of nuclear  pore protein and intermediate filament proteins of the nuclear lamina (lamina=  network of fibrous proteins that underlies and stabilizes the nuclear envelope) - Chromosome segregation takes place after the duplicated chromosomes are  correctly attached to kinetochore proteins that interact with the microtubules of the mitotic spindle  

o The cell cycle control system initiates chromosome segregation only after the  duplicated chromosomes are correctly aligned on the mitotic spindle

- After nuclear envelope is disassembled and dissolved.

o Microtubules (mitotic spindle) have access to the duplicated chromosomes and  capture them.

 Spindle microtubules grab hold of the chromosomes at kinetochores protein  complexes that assemble on the centromere of each condensed chromosome during late prophase

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o At randomly probing microtubule encountering a kinetochore will bind to it, thereby  capturing the chromosome. This kinetochore will bind to it, thereby capturing the  chromosome.  

 This kinetochore microtubule links to the chromosome to a spindle pore. o Kinetochores on sister chromatids face in opposite directions so they attach to  microtubules from opposite poles of the spindle, so that each duplicated  

chromosome becomes linked to both spindle poles.  

 This attachment to opposite poles is called bi-orientation, generates tension

on the kinetochores, which are being pulled in opposite directions.  

 This tension signals to the sister kinetochores that they are attached correctly

and are ready to be separated.  

o Microtubules attach to the kinetochores on the centromeres of the chromosomes.  - Kinetochore  

o Are attached to the centromeres on the chromosomes.  

o Each duplicated chromosome has two kinetochores – one on each sister chromatid which face in opposite directions.  

o Kinetochores recognize the special DNA sequence present at the centromere: if this  sequence is altered, kinetochores fail to assemble and consequently, the  chromosomes fail to segregate properly during mitosis.  

- Once the nuclear envolope is dissolved  

- Biorientation of kinetochore attachment on sister chromatids  

o Microtubules extended from opposite spindle poles will attach to kinetochores on  sister chromatids.  

o Microtubules are formed in the cytosol.  

Chromosomes assist in the assembly of the mitotic spindle  - Chromosomes can stabilize and organize microtubules into functional mitotic spindles.  - In cells without centrosomes the chromosomes nucleate microtubule assembly, and motor  proteins then move and arrange the microtubules and chromosomes into a bipolar spindle.

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- In animal cells that have centromeres a bipolar spindle can still be formed in this way if  the chromosomes are removed.  

- In cells with centrosomes, the chromosomes, motor proteins, and centrosomes work  together to form the mitotic spindle.  

Spindle Equator

- During prometaphase, the duplicated chromosomes, now attached to the mitotic spindle,  begin to move and line up at the equator of the spindle.  

o A balanced addition and loss of tubulin subunits is also requires to maintain the  metaphase spindle.  

- Alignment at equator is halfway between spindle poles, forming the metaphase plate o Requires expansion and contraction of tubules to align half-way  

- Formation of metaphase plate defines the start of metaphase.

- Metaphase plate if formed when duplicated chromosomes are aligned halfway between  the two spindle poles.  

- Disrupting attachment (error) of kinetochore or sister chromatids will cause a shift towards one of the spindle poles

o As a result one sister cell will have more chromosomes and one sister cell will have  less chromosomes.

Sister chromatid separation  

- Cohesion are active and hold sister chromatids together during S phase as soon  as the DNA is replicated, later these proteins are degraded by separase  - Linkage between sister chromatids (cohesion) is broken by separase in anaphase  o Before anaphase, separase is inactive due to securin being bound to separase to  keep it inactive  

o During anaphase, APC regulates securin. When APC is active it adds ubiquitin group  to securin  

o Cohesins binds to sister chromatids to hold them together. The complex is  dissociated by separase which degrades cohesion.

- Begins with the breakage of the cohesin. This release allows each chromatid- now  considered a chromosome- to be pulled to the spindle pole to which it is attached. This  movement segregrates the two identical sets of chromosomes to opposite ends of the  spindle.  

- The cohesion linkage is destroyed by a protease called separase. Before anaphase,  protease is held in an inactive state by an inhibitory protein called securin.  

PHASE PROTEIN  CAUSE EFFECT

BEFORE ANAPHASE  Cohesin  Holds sister chromatids  together

To prevent anaphase

BEFORE ANAPHASE Securing  Is bound to separase  Keeps separase inactive  ANAPHASE APC Degrades securin Activates separase  

ANAPHASE Separase Dissociates cohesion  from sister chromatids 

Separates sister  chromatids 

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Chromosome segregation (during anaphase)

- Movement of Myosin 1, Myosin 2, and Kinesin motor proteins are all towards the plus end of the filament  

- There are two parts in anaphase. Anaphase A and Anaphase B

- Anaphase A- depolymerization of tubules. Chromosomes are pulled poleward o Through shortening of kinetochore microtubules (due to loss of tubulin subunits),  forces are generated at kinetochores to move chromosomes towards their spindle  pole.  

o Kinesin – works on interpolar microtubules to create movement of the microtubules  - Anaphase B- movement of spindle poles  

o A sliding force is generated between interpolar microtubules from opposite poles to  push the poles apart.

o A pulling force acts to pull the poles toward the cell cortex thereby moving the two  poles apart.

o Dynein – acts on spindle poles

 Is a motor protein which moves the spindle poles.

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Nuclear envelope formation  

- The nuclear envelope reassembles during telophase and mitotic spindles disassemble o Proteins are phosphorylated to reassemble the envelope. Assembly of envelope  results in the disassembly of the mitotic spindle.

o Protein synthesis and gene expression are shut down during telophase  

- Assembly of the nuclear envelope starts the process of DNA transcription. The genome  (that was condensed during prophase) is now decondensed.  

o Expansion of the genome results in activation of transcription.

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Cytokinesis  

- division of the cytoplasm  

- Begins in Anaphase but not completed until the formation of two daughter nuclei (end of  telophase)

- Depends on actin and myosin filaments which form the contractile ring

- Mitotic spindle determines plane of cytokinesis and its timing

o mitotic spindles give direction to where the cell must be divided. formation of the metaphase plate is the area at which actin and myosin filaments form  contractile ring to divide the cell into two daughter cells

- overlapping interpolar microtubules (present in anaphase and metaphase)

o these give the cell an idea of where to form contractile ring which divides the cytoplasm into two daughter cells.  

o so the mitotic spindles determine where the contractile ring will be form and  thus where the cytokinesis will take place

 in most cases, there is symmetric division  

 but in embryotic cells there is asymmetric division which causes the  two daughter cells to have different proteins and molecules that  

causes differentiation into different cell types.  

PHA

TARGET CAUSE EFFECT

SE 

Cdk Cyclin Cdk binds to cyclin to Cyclin-cdk complex

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become active phosphorylates key proteins in  

the cell that are required to  

initiate particular steps in the  

cell cycle.

APC M-cyclin Ubiquitin Off

Wee1 M-CDK Phosphorylates CDK Off 

Cdc26 M-Cdk Dephosphorylated M-cdk On

P27 G1-Cdk S-Cdk

Physically interacts Off

G1 Rb G1-Cyclin G1/S-Cyclin

If no extracellular signal by  mitogens, it turns off  transcription by binding to  transcription regulators  

Off

G1 G1- Cdk

S-Cdk

Rb  

(retinoblasto ma)

Phosphorylates Rb = inactive So transcription factors can  bind to DNA  

= production of proteins  = cell ready to go to next  phase  

ON

G1 P53 P21 gene Synthesize P21 Synthesize

G1 P21 G1/S-Cdk Binds to G1/S-Cdk to  

Off

inactivate them when DNA is  

damaged

S Cdc-6 ORC Bonds to it to call in On

Cdc 26

MCDK Dephosphorylates m-Cdk to  activate it = positive  

feedback loop  

On  

M-Cdk CDC25 On  

Ch. 17 – Cytoskeleton  

Key points  

Dynamic instability and Treadmilling, Kinesin and Dynein, Muscle contraction, structure of cytoskeleton  filaments, Flagella, Cilia

Overview  

- The cytoskeleton is built on a framework of three types of protein filaments  o Intermediate filaments  

o Microtubules  

o Actin filaments

- Functions  

o Muscle contractions

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o Motility  

Key theory  

- Cytoskeleton is composed of three types of protein filaments  

o Intermediate filaments, microtubules, and actin filaments  

- Each type of filament has distinct mechanical properties  

- Each type of filament is composed of unique proteins subunits assembled via  polymerization  

Types of filaments  

NAME MONOMERS/  SUBUNITS  

FOUND STRUCTURE AND FUNCITON

INTERMEDIAT E FILAMENTS

Fibrous  

intermediate  filament proteins  

Cytosol, and around the nucleus  (nuclear lamina)

Are rope like fibers. Are flexible but very strong  

MICROTUBULE S

Tubulin Have one end attached to a single  microtubule-organizing center  

(centrosome)

Long hollow cylinders

Organize the location of  different organelles. More rigid  than the others and rupture  when stretched  

ACTIN  

FILAMENTS

Helical polymers  of the protein  actin  

Most highly concentrated in the  cortex (just beneath the plasma  membrane) but are dispersed  throughout the cell  

Flexible two stranded filaments – structural role  

Muscle contraction  

Intermediate filaments – formation  

- Cytoskeletal filaments found in the nucleus are intermediate filaments  - Intermediate filaments are like ropes made of long, twisted strands of protein.  - The strands of an intermediate filament is made of the subunit, fibrous  intermediate filament proteins  

- Intermediate filaments protect cells from mechanical stress because they have  high tensil strength and resist stretching  

- Intermediate filaments can connect cells at cell-cell junctions called  desmosomes  

- Each filament is made of eight strands, and each strand is made from staggered tetramers linked end to end  

o These subunits contain a central elongated rod domain with distinct unstructured  domains at either end  

o Intermediate filaments are the toughest most durable of the three types of  cytoskeletal filaments and can even survive treatment with concentrated salt  solutions and detergents  

o The rod domain consists of an extended alpha-helical region that enables pairs of  intermediate filament proteins to form stable dimers by wrapping around each other in an alpha-helical coiled-coil conformation  

- Alpha helical coiled-coil conformation  

o Two coiled-coil dimers, running in opposite directions, associate to form a staggered  tetramer.  

o Pairs of monomers associate to form a dimer  

 And two dimers then line up to form an antiparallel tetramer  

o These dimers and tetramers are the soluble subunits of intermediate filaments  o The tetramers assemble to generate the final rope like intermediate.

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 Tetramers can pack together into a helical array containing eight tetramer  strands  

 Which in turn assemble into the final rope like intermediate filament.  

- The central rod domains of different intermediate filaments are all similar in size and amino acid sequence  

o So that when they pack together they always form filaments of similar diameter and internal structure.  

o But the terminal domains vary greatly in size and sequence from one type of  intermediate filament protein to another.  

Classes of intermediate filaments  

- Intermediate filaments contain monomers with alpha-helical regions  - Intermediate filaments are most common in cells that are subject to mechanical stress o In these cells, intermediate filaments distribute the effect of locally applied forces to keep cells and their membranes from tearing in response to mechanical shear. - Intermediate filaments can be separated into four classes

o Filaments in each class are formed by polymerization of their corresponding  intermediate filament subunits  

- Keratin filaments  

o The most diverse class

o Every kind of epithelium in the vertebrate body has its own distinctive mixture of  keratin proteins.  

o Typically span the interiors of epithelial cells from one side of the cell to the other,  and filaments in adjacent epithelial cells are indirectly connected through  

desmosomes.  

o The ends of the keratin filaments are anchored to the desmosomes and the  filaments associate laterally with other cell components through the globular head  and tail domains that project from the surface  

o Many intermediate filaments are further stabilized by accessory proteins such as  plectin that cross-link the filaments and to adhesive structures in the desmosomes.  - The nuclear envelope is supported by a meshwork of nuclear lamina  o Nuclear lamins are intermediate filaments that are found in the nuclei of all animal  cells as well as plant cells  

o Line and strengthen the inside surface of the inner nuclear membrane are organized as two dimensional meshwork.  

o Constructed from intermediate filament proteins called lamins  

 So the nuclear lamina is made up of a specific class of intermediate filaments  called lamins  

o The nuclear lamins disassembles and reforms at each cell division, when the nuclear envelope breaks down during mitosis and re-forms in each daughter cell  

o Cytoplasmic intermediate filaments also disassembles in mitosis  

o This disassembly and reassembly is controlled by the phosphorylation and  dephosphorylation of the lamins.  

 Phosphorylated by kinases = weakens the binding between the lamin  

tetramers and causes the filaments to fall apart  

 Dephosphorylation by phosphatases at the end of mitosis cause the lamins to reassemble

Type

Intermediate  

filaments

Have great tensile strength They are the most durable  and toughest filaments of  the cytoskeletal filaments.

Form a network throughout the  

cytoplasm, surrounding the nucleus and  extending out to the periphery. There they are  anchored to the plasma membrane at cell-cell

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junctions called desmosomes where the  membrane is connected to that of another cell. Also found in the nucleus where they form a  meshworl called nuclear lamina. This underlies  and strengthens the nuclear envelope.

Class of  

intermediate  

filaments

What they form

Where  

Function

they’re  

found

Keratin filaments

Epithelial cells

Cytoplasm

Vimentin and  

vimentin-related  filaments

Connective tissue cells,  muscle cells, and  

supporting cells of the  nervous system (glial cells)

Cytoplasm

Neurofilaments

Nerve cells

Cytoplasm

Nuclear lamins

Strengthen the nuclear  envelope.

Nucleus Line the inner face of the  nuclear envelope and provide  

attachment sites for the  

chromosomes

Microtubules – overview  

- Can rapidly disassemble in one location and reassemble in another location.  - Mainly responsible for 

o transporting and positioning membrane-enclosed organelles within the cell  o and for guiding the intracellular transport of various cytosolic macromolecules  - Grow out from the centrosome – extend towards the cell periphery and create a system of  tracks within the cell, along which vesicles, organelles and other cell components can be  transported.  

- Role in cell division  

o When the cell enters mitosis, the microtubules disassemble and then reassemble  into the mitotic spindle.  

 The spindle provides the machinery that will segregate the chromosomes  equally into two daughter cells just before the cell divides.  

- Form stable structures: cilia and flagella  

o These are hair like structures

o Microtubules are stabilized by association with motor proteins that power the mobile appendages

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Microtubules – formation  

- Microtubules are cytoskeletal structures that provide tracks for

guiding intracellular transport of vesicles, organelles and other cell

components in the cytosol  

- Vesicles and organelles are transported along microtubules in the

cytosol  

- Built from molecules of tubulin – each molecule is composed of two

similar globular proteins called alpha and beta tubulin  

o These are bound together by noncovalent bonding to form the

wall of the hollow cylindrical microtubule.  

- the tubelike structure if made of  

o 13 parallel protofilaments each a linear chain of tubulin dimers

with alpha and beta tubulin alternating along its length  

o Alpha tubulin sticks up at one end (positive) and beta tubulin

sticks up at the other (negative) = polarity  

- Protofilament

o Give the microtubule structural polarity  

 Have a positively charges and negatively charged end.  

o This polarity is necessary for the assembly of microtubules and for their job once  formed  

o If the microtubules did not have polarity they could not guide intracellular transport  (example)

The centrosome  

- Is the major microtubule-organizing center in animal cells  

- Microtubules grow from centrosomes inside the cell. The centrosome controls the location,  number, and orientation of the microtubules.  

o In animal cells the centrosome organizes the microtubules to extend through the  cytoplasm  

- Each microtubule grows and shrinks independently of its neighbors.  

o The microtubules anchored in a centrosome is continually changing (new tubules  grow and old microtubules shrink)

- The centrosome consists of a pair of centrioles 

o The centrioles are surrounded by a matric of proteins

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- The centrosome matric includes a lot of gamma-tubulin  

o Each gamma-tubulin ring complex serves as the starting point nucleation site for  the growth of one microtubule.  

o The alpha/beta-tubulin dimers add to each gamma-tubulin ring complex in a specific orientation  

 The result is that the minus end of each microtubule is embedded in the  centrosome  

 and growth occurs only at the plus end that extends into the cytoplasm  

Centrioles 

- each centriole is paired to another. The paired centrioles are at the center of the  centrosome  

- they sit perpendicular to each other and are made of a cylindrical array of short  microtubules.  

- Centrioles have no role in nucleation of microtubules from the centrosomes (don’t help  them grow)

o They act as the organizing centers for the microtubules in cilia and flagella   Here they are called basal bodies

Dynamic instability  

- Growing microtubules display dynamic instability.  

- Once a microtubule has been nucleated,  

o it grows outwards from the organizing center due to the addition of the alpha-beta tubulin dimers to the plus end.  

o Then the microtubule can undergo a conformational change that will cause it to  shrink rapidly inward by losing tubulin dimers from its free plus end.  

 It may shrink partially and then start growing again.  

 Or it can disappear completely to be replaced by a new microtubule that  grows from the same gamma-tubulin ring complex  

- This behaviour is called dynamic instability  

o It allows the microtubules to undergo rapid remodeling and is crucial for their  function  

- Commonly, the microtubule that is growing out of the centrosome begins to disassemble  o To prevent it from disassembling, it’s positively charged end can be attached to  another molecule or cell structure in a more distant region of the cell.  

o The microtubule will then establish a relatively stable link between that structure  and the centrosome.  

- Overview

o An alpha/betta-tubulin dimer is added to the plus end of a microtubule extending  across the cytoplasm  

o The growing microtubule displays dynamic instability and can rapidly disassemble  due to the instability  

o To stabilize the growing microtubule its positive end can be attached to another cell  structure or molecule  

o The microtubule thus forms a stable link between the centrosome and a cell  structure/ molecule.  

o And therefore, the microtubule must attach to something in order to remain  assembled

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Dynamic instability and GTP 

- Dynamic instability is driven by GTP hydrolysis  

- GTP hydrolysis and whether GTP or GDP is bound to tubulin is an important mechanism to  control the dynamic instability of microtubules.  

o Growing microtubules is a process used for visualizing the dynamic instability  because EB1 binds to the GTP-tubulin cap on microtubules  

- The dynamic instability of microtubules is due

to the intrinsic capability of tubulin dimers to

hydrolyze GTP.  

o Each free tubulin dimer contains one

GTP molecule, tightly bound to beta

tubulin

 Tubulin dimers containing GTP

bind more tightly together than

do tubulin dimers carrying GDP  

 Therefore, rapidly growing plus

ends of microtubules (which have

the added tubulin dimers with

GTP bound) tend to keep growing

Dynamic instability and GDP 

- When the microtubule growth is slow, the b

tubulin becomes depolymerized and the

microtubule shrinks  

o Beta-tubulin hydrolyses the GTP  GDP

shortly after the dimer is added to a

growing microtubule.  

o The GDP remains tightly bound to the

beta-tubulin  

 If microtubule growth is slow, the

dimers in the GTP cap will

hydrolyze their GTP GDP before

the new dimers with more GTP

have time to bind  

 Therefore the GTP cap is lost

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o Because the GDP carrying dimers are less tightly bound in the polymer, the  protofilaments peel away from the plus end and the dimers are released  

o Thus the microtubule shrinks  

- The GDP-tubulin that is released joins the unpolymerized tubulin already in the cytosol.  - They then exchange their GDP for GTP and become ready to be added to another  microtubule that is in growth phase  

Drugs modify microtubules  

- Drugs that prevent the polymerization and depolymerization of tubulin dimers can have  rapid and profound effects on the organization fo the microtubules – thereby on the  behaviour of the cell.  

- Example: mitotic spindle in mitosis

o If a cell in mitosis is exposed to colchicine  

 Colchicine binds tightly to free tubulin dimers and prevents their  

polymerization into microtubules

o The mitotic spindle rapidly disappears and the cell stalls in the middle of mitosis  Because it is unable to divide its sister chromatids because there are no  mitotic spindle (made from microtubules) attached to the sister chromatids  because the drug prevented the beta-tubulin from being polymerized and  

thus growing the mitotic spindle.  

o When cells are treated with colchicine both the ER and the Golgi change their  location.  

 The ER (connected to the nuclear envelope) collapses around the nucleus;  the Golgi fragments into small vesicles, which then disperse throughout the  cytoplasm.  

o When colchicine is removed, the organelles return to their original positions,  dragged by motor proteins moving along the re-formed microtubules.  

- Example: the drug taxol

o Binds tightly to microtubules and prevents them from being depolymerized (losing  beta-tubulin subunits)  

o Because new subunits can still be added, the microtubules can grow but not shrink  This causes the cell to arrest in mitosis  

- The inactivation and destruction of spindle eventually kills the cell.

- But this can be used in treatments of cancers because by using microtubule-stabilizing or  -destabilizing antimitotic drugs the cell can not divide and thus dies  

Motor proteins – drive intracellular transport  

- When the cell enters mitosis, the cytoplasmic microtubules disassemble and then  reassemble into an intricate structure called the mitotic spindle.  

- Microtubules can also form stable structures such as the cilia and flagella. These  structures extend from the surface of cells.

- Each protofilament has structural polarity, with alpha-tubulin exposed on one end and  beta-tubulin exposed at the other, and this polarity is the same for all protofilaments,  giving structural polarity to the microtubule  

o The beta-tubulin is called its plus end and the alpha-tubulin the minus end. Without  polarity, microtubules could not guide intracellular transport.  

- Saltatory movements can our along either microtubules or actin filaments. In both cases,  the movements are driven by motor proteins, which use the energy derived from ATP  hydrolysis to travel in a single direction.

Motor protein

Move along what and in  what direction

Structure

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Kinesins

Generally move along  cytoplasmic microtubules (outwards from the cell  body)

Has two globular  

heads at one end,  

which bind and  

hydrolyse ATP and  interact with  

microtubules.

And a single tail which interacts with cargo

Dynein’s

Move toward the minus  end (toward the cell  

body)

Has two globular  

heads at one end,  

which bind and  

hydrolyse ATP and  interact with  

microtubules

And a single tail which interacts with cargo

Cytosolic dynein’s attached to the  Golgi membrane  pull the Golgi  

along  

microtubules in  the opposite  

direction, inwards  towards the  

nucleus.

Motor protein families: Dynein and Kinesins  

- Kinesins  

o Move towards the plus end of the microtubule (outward from the cell body) o Kinesins attached to the outside of the ER membrane pull the ER outward along  microtubules  

o kinesins move towards the plus end of microtubules  

- Dynein  

o Cytoplasmic dynein transport cargo towards the minus ends of microtubules and are responsible for backward transport in axon microtubules.,  

o Move towards the negatively charged end of the microtubule (towards the cell body) o Are attached to the Golgi membrane to pull the Golgi apparatus along microtubules  in the opposite direction (towards the nuclear)

o In anaphase B, dynein motor proteins work on spindle poles  

o Movement of Dynein causes flagellum to bend  



Different motor proteins transport different cargo  

- A cargo is being transported towards the minus end by a motor protein, it must  be interacting with microtubules (cytoskeletal filament)  

- Both kinesins and dynein are generally composed of two dimers (subunits) that have  globular ATP-binding heads and a single interacting tail  

o The heads interact with microtubules in a stereospecific manner, so that the  motor protein will attach to a microtubule in only one direction.  

o The tail of the motor protein binds stable to some cell component, such as an  organelle or vesicle, and thus determines the type of cargo that the motor protein  can transport. 

- The globular heads are enzymes with ATP-hydrolyzing (ATPase) activity  o This reaction provides the energy for driving a directed series of conformational  changes in the head to enable it to move along the microtubule by a cycle of  binding, release, and rebinding to the microtubule.  

- Most kinases move toward the plus end of a microtubule, whereas dyneins move toward  the minus end of the microtubule.

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- Both types of microtubule motor proteins exist in many forms

o Each transports a different type of cargo.  

o The tail of a motor protein determines that cargo the protein transports.  - Organelles move along microtubules.  

- Examples:

o the endoplasmic reticulum aligns on microtubules as kinesins pull it away from the  nucleus and stretch it out

o Dyneins pull the Golgi apparatus towards the nucleus to regulate the area between  internal membranes

Cilia and flagella contain stable microtubules moved by Dynein  -

Actin filaments are thin flexible and highly dynamic  

- Actin filaments are polymers of the protein actin. Are present in all eukaryotic cells and are essential for many of the cells movements, especially those involved around the cell  surface.  

- Many actin filaments are unstable. They can also form stable structures in cells (like the  contractile apparatus of muscle cells)  

o Actin filaments interact with a large number of actin-binding proteins that enable filaments to serve a variety of functions in cells.  

o Depending on which of these porin’s they associate with, actin filaments can form  stiff and stable structure.  

 Can form microvilla  

 They can form small contractile bundles that can contract and act like tiny  muscles  

 They can form temporary structures  

 Or contractile ring that pinches the cytoplasm in tow  

o Actin-dependant movements usually require actins association with myosin (a motor protein)

- Actin filaments allow animal cells to adopt a variety of shapes, and perform a variety of  functions

Treadmilling  

- Actin can grow by addition of actin monomers at either end. But the rate of growth Is  faster at the plus end.

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- The ‘naked’ actin filament is unstable and it can disassemble from both ends.  - The actin filament is a twisted chain of actin moleules  

- Free actin monomers carry ATP  

o The actin monomer hydrolyses its bound ATP ADP soon after being incorporated  with the actin filament  

o Hydrolyses of ATP reduced the strength of binding between the monomers =  decreases the stability of the growing polymer  

- If the concentration of free actin is high  

o An actin filament will grow rapidly, adding monomers at both ends  

- Intermediate concentrations  

o Actin monomers add to the plus end at a faster rate than the ATP can be hydrolyzed so the plus end grows.  

o At the minus end ATP is hydrolyzed faster than the new monomers can be added =  the filament loses subunits from its minus end at the same time they are added to  the plus end. This is called treadmilling

- An actin filament undergoing treadmilling at the leading edge of a  lamellipodium remains the same size.  

- When ATP-actin adds to the plus end of an actin filament at the same rate that  ADP-actin is lost from the minus end, treadmilling occurs  

- Treadmilling involves simultaneous gain of monomers at the plus end of the actin filament  and loss at the minus end  

o When the rates of addition and loss are equal, the filament remains the same size.  o Dynamic instability involved a rapid switch from growth to shrinkage at only the  plus end of the microtubule.  

o As a result, microtubules tend to undergo more drastic changes in length than actin  filaments.  

- During dynamic instability  

o GTP-tubulin adds to the plus end of a growing microtubule

o When FTP-tubulin addition is faster than GTP hydrolysis, a GTP cap forms at that end o When the rate of addition slows, the GTP cap is lost, and the filament experiences  catastrophic shrinkage via loss of GDP-tubulin from the same end.

o The microtubule will shrink until the GTP cap is regained – or until the microtubule  disappears.  

- Actin filaments can undergo treadmilling where actin monomers in the cytosol that carry  ATP can joing to the filament. The ATP is hydrolyzes to ADP soon after assembly into a  growing filament. The ADP molecules remain trapped within the actin filament, unable to  exchange with ATP until the actin monomer that carried them dissociates from the  filament.

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Myosin 1 

- Myosin attaches to actin; ATP binding reduces the affinity of myosin for actin; myosin is  cocked as its head is displaced along the actin filament; the power stroke puts myosin in a  rigor conformation  

- All actin-dependant motor proteins belong to the myosin family. They bind to and  hydrolyze ATP, which provides energy for their movement along actin filaments toward the plus end.

- Myosin 1 is simpler in structure than myosin 2 and is present in all types of cells  - Myosin 1 molecules  

o Have a head domain and a tail  

o The single globular head domain binds to an actin filament and has the ATP hydrolyzing motor activity that enables it to move along the filament in a repetitive  cycle of binding, detachment, and rebinding  

o The tail varies among the different types of myosin-I and determines what type of  cargo the myosin drags along.  

 For example, the tail may bind to a particular type of vesicle and propel it  through the cell along actin filament tracks  

 Or It may bind to the plasma membrane and pull it into a different shape  - Myosin I is the simplest myosin. It has a single globular tail that attaches to an atin  filament and a tail that attaches to another molecule or organelle in the cell.  o This arrangement allows the head domain to move a vesicle relative to an actin  filament, which in this case is anchored to the plasma membrane.  

o Myosin-I can also bing to an actin filament in the cell cortex, ultimately pulling the  plasma membrane into a new shape.  

o The head group always walks toward the plus end of the actin-filament.  

Actin and myosin 1 

- Myosin is attached to actin before ATP binds; ATP binding reduces the affinity of the head  for actin and allows it to let go of the filament. ATP hydrolysis, and the subsequent release  of inorganic phosphate, triggers the power stroke that returns myosin to its original rigor  conformation. At the end of this cycle, the myosin head has moved to the new position on  the actin filament and is prepared for the next cycle.  

- myosin and other actin-binding proteins regulate the location, organization, and behavior  of actin filaments.  

- the activities of these proteins are controlled by extracellular signals

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o allowing the cell to rearrange its actin cytoskeleton in response to the environment.  - The extracellular signal molecules that regulate the actin cytoskeleton activate  a variety of cell-surface receptor proteins,  

o These cell-surface receptor proteins in turn activate various intracellular signaling  pathways.  

o These pathways often converge on a group of closely related monomeric GTPase proteins called the Rho protein family.  

- activation of different members of the Rho family affects the organization of actin  filaments  

Myosin 2 

- Can associate with one another to form myosin filaments  

- A molecule of myosin-II contains two identical heavy chains, each with a globular head and extended tail.  

o It also contains two light chains bound to each head.  

o The tails of the two heavy chains form a single coiled-coil tail  

o The coiled-coil tails of myosin-II molecules associate with one another to form a  bipolar myosin filament in which the head project outward from the middle in  opposite directions.

- Myosin-II tails can associate to form long filaments with the globular heads projecting  outwards in opposite directions. The bare region in the middle is only tails - The bipolar nature of myosin filaments allows for the “sliding” of actin filaments with  which it associates.  

o This is the fundamental mechanism behind muscle contraction  

Myosin filaments 

- Muscle myosin belongs to the myosin-II subfamily (Are all dimers with two globular ATPase  heads at one end and a single coiled-coil tail at the other.  

- Clusters of myosin-II molecules bind to each other through their coiled-coil tails, forming a  bipolar myosin filament from which heads project  

o The myosin filament has two sets of myosin heads pointing in opposite directions so that one set binds to the actin filaments in one orientation and moves the filaments  one way and the other set binds to the actin filaments and moves the filaments in  the other direction.

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Actin filaments slide against myosin filaments during muscle  contraction  

- The nuclei of the contributing cells are retained in the muscle fiber and lie just beneath the plasma membrane. The bulk of the cytoplasm is made up of myofibrils, the contractile  elements of the muscle cell.

- A myofibril consists of a chain of identical tiny contractile units called sacromeres o Highly organized assemblies of actin and myosin filaments composed of muscle  specific form of myosin-II  

o The myosin filaments are centrally positioned at each sarcomere, whereas the actin  filaments/ thin filaments extend inward from each end of the sarcomere where they  are anchored by their plus ends to the Z disc  

o The minus ends of the actin filaments overlap with the ends of the myosin filaments o As a result a myosin filament slides sets of oppositely oriented actin filaments past one another.  

- The bundle of actin and myosin filaments generates a strong contractile force.  There are small bundles called contractile bundles that assemble in the contractile ring  that pinches a dividing cell in two.  

- The contraction of a muscle cell is caused by a simultaneous shortening of all the cell’s  sarcomeres, which is caused by the actin filaments sliding past the myosin filaments, with  no change in the length of either type of filament.  

- The sliding motion is generated by myosin heads that project from the sides of the myosin  filament and interact with adjacent actin filaments

- When a muscle is stimulated to contract, the myosin heads start to walk along the actin  filament in repeated cycles of attachment and detachment. During each cycle, a myosin  head binds and hydrolyzes one molecule of ATP.  

o This causes a series of conformational changes that move the tip of the head along  the actin filament

o Causing the myosin heads to pull against the actin filament causing it to slide  against the myosin filament.  

o The concerted action of myosin heads pulling the actin and myosin filaments past  each other causes the sarcomere to contract.  

Skeletal muscle fibers  

- Are large, multinucleated individual cells formed by the fusion of many separate smaller  cells.  

- The nuclei of the contributing cells are retained in the muscles fiber and lie just under the  plasma membrane.  

- Most of the cytoplasm is made up of myofibrils, the contractile elements of the muscle  cell.  

- A myofibril consists of a chain of identical tiny contractile units, or sarcomeres.  o These are highly organized assemblies of actin and myosin filaments composed of a o ]=muscle-specific form of myosin-II

- The myosin filaments are centrally positioned in each sarcomere, the more slender actin  filaments extend inward from each end of the sarcomere where they are anchored by their plus ends to a structure called the Z disc  

- The contraction of a muscle cell is caused by a simultaneous shortening of all the cell’s  sarcomeres, which is caused by the actin filaments sliding past the myosin filaments, with  no change in the length of either type of filament  

o The sliding motion is generated by myosin heads that project from the sides of the  myosin filament and interact with adjacent actin filaments

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Muscle contraction  

Interacting filaments of actin and myosin  

- Myosin-II molecules can associate with one another to form myosin filaments  o A molecule of myosin-II consists of two identical heavy chains each with a globular  head and an extended tail.  

o The tails of the two heavy chains form a single coiled-coil tail.  

o The coiled coil tails associate with one another to form a bipolar myosin filament in  which the heads project outward from the middle in opposite directions  

- The myosin-II in non-musical cells is also activated by a rise in Ca2+ but the mechanism of activation is different from that of the muscle-specific myosin-II which alters the myosin  conformation and enables it to interact with actin. A similar activation mechanism  operated in smooth muscle. The mode of myosin activation is slow, because time is  needed to enzyme molecules to diffuse to the myosin heads and carry out the  phosphorylation and subsequence dephosphorylation.  

- However, the mechanism can be activated by a variety of extracellular signals.  Muscle contraction is triggered by a sudden rise in cytosolic Ca2+ - The force generating molecular interaction between myosin and actin filaments take place only when the skeletal muscle received a signal from a motor nerve. The neurotransmitter  released from the nerve terminal triggers an action potential in the muscle cell plasma  membrane. The electrical excitation spreads quickly into a series of membranous tubes  called transverse tubules or T-tubules that extend inward from the plasma membrane  around each myofibril  

- The electrical signal is then relayed to the sarcoplasmic reticulum, an adjacent sheath of  interconnected flattened vesicles that surrounds each myofibril  

- The sarcoplasmic reticulum is a specialized region of the endoplasmic reticulum in muscle  cells. It contains a very high concentration of Ca2+ and in response to the incoming  electrical excitation, the Ca2+ is released into the cytosol through ion channels that open  in the sarcoplasmic reticulum membrane in response to the change in voltage across the  plasma membrane and T tubules  

- In muscle cells the rise in cytosolic Ca2+ concentration activated a molecular switch made of specialized accessory proteins closely associated with the actin filaments. One is  trypomysin. A ridged, rod-shaped molecule that binds in the groove of an actin filament.  

- The other is troponin a protein complex that includes Ca2+ sensitive protein associated  with the end of a tropomysin molecule.  

- When the concentration of Ca2+ rises in the cytosol. Ca2+ binds to troponin and induces a change in the shape of the toponin complex. This in turn causes the trypomysin molecules  to shift their positions slightly, allowing myosin heads to binds to actin filaments, initiating  contraction.

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The big picture  

- Muscles contract by a sliding filament mechanism  

- A) the myosin and actin filaments of a sarcomere overlap with the same relative polarity  on either side of the midline.  

o The actin filaments are anchored by their plus ends to the Z disc and myosin  filaments are bipolar

- B) during contraction, the actin and myosin filaments slide past each other without  shortening. This sliding motion is driven by the myosin heads walking towards the plus  end of the adjacent actin filaments.

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77

Ch. 20 - Tissue maintenance and revival  

Main points  

Causes of genome instability, reproductive cloning, virus infection causing cancer, Malignant vs Benign tumor,  mutagen, key pathways in cancer, events leading to oncogene formation and tumor suppressor, p53, Ras,  cadherin, telomerase, Why cancer cells are advantageous to normal cells? Fibronectin, Tight Junction, GAP  junction, desmosome, hemidesmosome, adherin junction, basal lamina, polysaccharide function

Tissue 

- In multicellular organism cells are organized into Tissues  

- Extracellular matrix  

o Extracellular molecules secreted by the cell, providing support and structure  - Cell junctions

o Links cells together  

Animal tissue 

- Connective  

o Can be tough and flexible

o Hard and dense  

o Or soft and transparent  

- Nervous

- Muscular  

- Epithelial  

Connective tissues  

Animal connective tissues consist largely of extracellular matrix  - Extracellular matrix carries the mechanical load  

Collagen  

- The tensile strength is chiefly provided by a fibrous protein called collagen.  - Collagen is a type of protein that has many varieties.  

- The various types of connective tissues owe their specific characters to the type of  collagen they contain  

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Elastin  

- Specialized  

- polysaccharide molecules  

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collagen  

- It is a long stiff, triple-stranded helical structure, in which three collagen polypeptide  chains are wound around one another in a rope-like helix, then to fibrils and finally fibers  Collagen fibrils  

- Some types of collagen molecules in turn assemble into ordered polymers called collagen  fibrils  

- These are thin cables and many micrometers long  

- These can pack together into thicker collagen fibers.  

- Other types of collagen molecules decorate the surface of collagen fibrils and link the  fibrils to one another and to other components of the extracellular matrix  Collagen fibers  

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Fibroblasts  

- The connective-tissue cells that manufacture procollagen and inhabit the extracellular  matrix in the skin, tendons, and many other connective tissues  

Osteoblasts  

- In bone the connective-tissue cells that manufacture procollagen and inhabit the  extracellular matrix are called osteoblasts

Procollagen

- Is the precursor form of collagen with additional polypeptide extensions at each end that  obstruct premature assembly into collagen fibrils.  

- Almost all of the molecules synthesized by the fibroblasts and osteoblasts are synthesized  intracellularly and then secreted by exocytosis.

o Outside the cell they assemble into huge cohesive aggregates. If assembly were to  occur inside the cell, the cell would become choked with these products.  

- The cells avoid this by secreting procollagen  

- Extracellular enzymes called procollagen proteinases cut off the terminal extensions on  procollagen to allow assembly only after the molecules have emerged into the  extracellular space.  

Cells organize the collagen they secrete  

- Collagen fibrils must be correctly aligned to function properly. The connective-tissue cells  that produce collagen control its orientation by  

1. Depositing the collagen in an oriented fashion  

2. Rearranging it  

- During development, fibroblasts work on the collagen they have secreted by moving over  it and pulling on it.  

o This helps to compact the collagen into sheets and draw it out into cables.  - This mechanical role of fibroblasts in shaping collagen matrices occurs by the fibroblasts  migrating out from the explants along the aligned collagen fibers. Thus, the fibroblasts  influence the alignment of the collagen fibers, and the collagen fibers in turn affect the  distribution of the fibroblasts  

Cell attachment  

- In order for cells to pull on the matrix, they must attach to it but cells do not attach to bare collagen. Fibronectin provides a linkage

- Part of the fibronectin molecule binds to collagen while another part forms the attachment  site for a cell.  

Integrin  

- A cell attaches itself to fibronectin by means of a receptor protein called an integrin.  - Extracellular and intracellular signals can activate integrin which spans the cells plasma  membrane  

- When the intracellular domain of the integrin binds to fibronectin, the intracellular domain binds (through a set of adaptor molecules) to an actin filament inside the cell.  o Without this internal anchor to the cytoskeleton, integrins would be ripped out of  the lipid bilayer as the cell attempted to pull itself along the matrix.  

- The formation and breakage of the attachments on either end of an integrin  molecule allow the cell to crawl through a tissue, grabbing hold of the matrix at its front  end and releasing its grip at the rear.  

- Integrins coordinate this catch and release by undergoing conformational changes.  Binding to a molecule on one side causes the integrin molecule to stretch out into an  extended, activated state so that It can latch onto a different molecule on the opposite  end.

- An intracellular signaling molecule can activate the integrin from the cytosolic side,  causing it to reach out and grab hold of an extracellular structure

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- binding to an extracelllar structure can switch on intracellular signalling pathways by  activating protein kinases that associate with the intracellular end of the integrin. In this  way the cells external attachments regulate whether it lives, dies and whether  it grows, divides, or differentiates

Polysaccharides  

- gels of polysaccharides and proteins fill spaces and resit compression  

Glycosaminoglycan (GAGs) resist compression  

- are built from GAG  

- are negatively charged polysaccharide chains made from repeating disaccharide units.  - Are covalently linked to core proteins to form proteoglycans

o Diverse in size, shape, and chemistry  

- Extremely hydrophilic  

o due to the negative charge on the repeating disaccharide units  

o tend to adopt highly extended conformations which occupy a large volume of space  relative to their mass.

o Their negative charges attract cations that are osmotically active, causing large  amounts of water to be sucked into the matrix. This gives rise to swelling  pressure, which is balanced by the tension in the collagen fibers interwoven with  the proteoglycans.  

- provide hydrated spaces around the cell

- Bind to secreted growth factors and other proteins that serve as extracellular signals for  cells  

- Regulate cell migration  

Epithelial cells  

- There are many different types of cells in the body, the majority of which are organized  into epithelia.  

- Epithelia are multicellular sheets in which cells are joined together side to side.  - Cells joined together into an epithelial sheet create a barrier

Apical  

- Exposed to fluids

Basal  

- Attached to a sheet of connective tissues – basal lamina

- The basal lamina consists of a thin tough sheet of extracellular matrix, composed mainly  of a specialized type of collagen and a protein called laminin

- Laminin  

o Provides adhesive sites for integrin molecules in the basal plasma membrane of  epithelial cells and it thus serves a linking role  

Cell junctions  

Tight junctions  

- Make a tight seal between neighboring cells to prevent the leakage of water-soluble  molecules across the epithelium through the gaps between the cells – helps polarize cells  - Also play a key role in maintaining the polarity of the individual epithelial cells in two ways  1. The tight junctions around the apical region of each cell prevents diffusion of  proteins within the plasma membrane and so keeps the apical domain of the  plasma membrane different from the basal domain  

2. In many epithelia the tight junctions are sites of assembly for the complexes of  intracellular proteins that govern the apico-basal polarity of the cell interior

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Claudins and  

occluding

Junctions providing mechanical strength  

Adherens junctions  

- Cadherin connected to actin filaments  

- Adherens junctions and desmosomes are both built around cadherin transmembrane  proteins  

- Cadherin molecule in the plasma membrane of adjacent cells bind to each other  extracellularly

- Inside the cell cadherin molecules are attached via linker proteins to actin (cytoskeletal)  filaments  

- Form a continuous adhesion belt around each of the interacting epithelial cells  - This belt is located near the apical end of the cell.  

- This network of actin filaments can contract giving the epithelial sheet the capacity to  develop tension and to change its shape.  

o By shrinking the apical surface along one axis the sheet can roll itself into a tube  o By shrinking its apical surface locally along all axes at once, the sheet can fold into  a cup and create a spherical vesicle by pinching off from the rest of the epithelium.  Desmosome

- Joins the intermediate filaments in one cell to those in its neighbor  

- Cadherin connects to keratin filaments

- A different set of cadherin molecules connects to keratin filaments- the intermediate  filaments found specifically in epithelial cells.  

- Bundles of keratin filaments cross the cytoplasm and are welded to a spot via desmosome  junctions to the bundles of keratin filaments in adjacent cells.  

- This arrangement gives tensil strength on the epithelial sheet and is characteristic of  tough exposed epithelia such as the epidermis of the skin  

Hemidesmosome  

- Anchors intermediate filaments in a cell to the basal lamina  

- Attachment of integrin to laminin in basal lamina and keratin filaments inside the cell  - The extracellular domains of the integrins bind to lamina in the basal lamina; inside the  cell, the integrin tails are linked to keratin filaments creating a structure.  

Gap junctions  

- Is the final type of epithelial cell junction

- Narrow gap spanned by the protruding ends of many identical transmembrane protein  complexes called connexons between two cell membranes that are parallel and close to  each other

Connexon  

- The connexons are aligned end-to-end to form a narrow water-filled channel across the two plasma membranes. The channels allow inorganic ions and small water-soluble molecules  to move directly from the cytosol of one cell to that of another.  

Stem cells 

Terminally differentiated cells: cells that need continual replacement and are  unable to divide  

- They lie at the dead end of their developmental pathway.  

Precursor cells: cells that replace terminally differentiated cells  - The cells that replace the terminally differentiated cells that are lost are generated from a  stock of proliferating precursor cells which derive from a smaller number of self-renewing  stem cells