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CLEMSON / Biology / BIOL / How can you tell if a protein has been extruded into a microsome from

How can you tell if a protein has been extruded into a microsome from

How can you tell if a protein has been extruded into a microsome from


School: Clemson University
Department: Biology
Course: Cell Biology
Term: Spring 2019
Tags: Biology, cellular biology, endomembrane system, actin, and myosin
Cost: 50
Name: BIOL 4610 Exam 2 Study Guide
Description: These notes cover what is expected to be on the next exam, if you have any questions or need clarifications please refer to the textbook!
Uploaded: 02/28/2019
21 Pages 15 Views 9 Unlocks

wdbrotherton (Rating: )

Eugene Bishop (Rating: )

Did not very helpful at all. Especially for the value, it did very little to help prepare me. I always build my own, and this was the one time I just did not have a chance. This was definitely not worth $30. For concepts like this, study guides that are just bulk text are only so helpful, without the images from the powerpoints tied into the study guide, it is just weak. Outside of that, the notes were good, just not useful.

Biology 4610 Exam 2 Study Guide Cellular Endomembranes and Protein Transport

How can you tell if a protein has been extruded into a microsome from an mrna encoding secretory protein?

∙ Overview of eukaryotic protein sorting

o Protein sorting can also be called protein trafficking or protein targeting o Proteins are made on cytosolic free ribosomes

o Protein remains in cytosol 

 unless signal/ target sequence directs otherwise 

o Polysome (mRNA+ ribosome) with signal sequence is transferred to rough ER  and enter the lumen. Then they travel to the Golgi where there are then released  and travel to the specific part of the cell

o Secretory pathway: not secreted, just travel from cytosolic free ribosome to the  ER 

∙ The Endoplasmic Reticulum

o Most eukaryotic cells have a very organized ER

o It is an enormous organelle, probably contains half the membrane in the cell.  o ER is a lacey organelle that is throughout the organelle

Where can the proteins go after they pass through the golgi?

o The inside of the ER is completely continuous 

 Lumen

o RER and SER are part of a single organelle, however the ER is made of a  majority of RER

 Dots are docked ribosomes on ER producing secretory proteins

 The proteins in the RER are different form the proteins present in the SER  Vesicles bud from the smooth ER and bind to the Golgi

o The ER is perinuclear, which means it forms a “cup” around the nucleus   Almost completely surrounding the nucleus

o Cell disruption produces ER­derived rough and smooth microsomes

 Homogenize the cell by any method causes the ER to fragment and reform into small vesicles called microsomes. 

 A rough microsome the interior is biochemically equivalent to ER Lumen ∙ all functions and proteins are  exactly the same as the internal 

How does the cell know which proteins to send to the lysosome?

If you want to learn more check out Does the upper extremities are not a part of the axial skeleton?

portion of the rough ER in the cell

 the rough and smooth microsomes can be purified through equilibrium  density centrifugation

∙ the smooth microsomes will be found in a band at the top or 

floating at the surface in the low concentration of sucrose, because 

they have a smaller density. These molecules have a smaller  Don't forget about the age old question of When was the coca cola company started?

density because there are no proteins at the surface.

∙ The rough microsomes have a higher density, which means they 

will be found towards the bottom of the tube in the high sucrose 


∙ The signal hypothesis

o The signal/ target sequence directs the polysome to dock at the rough endoplasmic reticulum. 

o The signal sequence reacts with the receptor on the surface of the ER, which  allows the polysome to bind. Once the polysome binds the growing polypeptide  chain is inserted into the lumen and the signal sequence is cleaved. Then, the fully synthesized protein will be entered into the ER lumen. 

o The experiment behind the discovery was performed by Blobel in the 1970s  mRNA encoding secretory protein was added to two different tubes: one  with microsomes and one without microsomes and allowed to complete in  vitro translation. The proteins from each tube were then ran on an SDS PAGE gel

 The tube without microsomes produced a protein that was larger than  normal.

 The tube with the microsomes produced a protein that was the correct size.  This data supports the signal hypothesis because the signal sequence adds  nucleotides, thus making it longer than normal. So, it must dock on ER  (microsomes) and have the signal sequenced cleaved in order to be the  correct size. 

o The signal peptide has multiple names: signal sequence, leader sequence, start transfer sequence We also discuss several other topics like Which sex is the homogametic sex in humans?

o There is no consensus sequence across all organisms, however there are specific  regions that are conserved

 Hydrophobic region, followed by positive and negative amino acids ∙ Translation and Translocation occur simultaneously in the cell

o A cell free protein synthesis was performed without the presence of microsomes,  then after the completed proteins with signal sequences were produced  microsomes were added Don't forget about the age old question of Does strategic management work for small as well as large firms?

 There was no incorporation of the mature protein into the microsome and  no removal of the signal sequence

o A cell free protein synthesis was performed in the presence of microsomes  A mature protein chain without the signal sequence was produced and  found within the microsome,

o Translocation is the movement of protein into the ER

∙ How can you tell if a protein has been extruded into a microsome from an mRNA  encoding secretory protein?

o N­terminal signal peptide is cleaved off by signal peptidase within the microsome o The protein is resistant to proteases because it is protected by the membrane the  protein is bound is

o The protein is not resistant to proteases in the presence of detergent because the  detergent will break up the membrane and allow the protease to enter, degrading  the protein

o The protein become glycosylated by enzymes within the microsome ∙ A signal recognition particle directs the signal peptide to receptors on the rough  endoplasmic reticulum 

o The SRP bridges the gap between the forming peptide and the receptor on the ER  membrane

o The molecule is a ribonucleicprotein composed of 6 proteins that are highly  branched RNA molecule

o SRP binds to the signal sequence and temporarily stops transcription. The  complex the docks the polysome onto the SRP receptor on the ER membrane. ∙ Translocation of soluble secretory proteins into the ER lumen Don't forget about the age old question of Define polyploidy.

o The polysome is guided to the receptor by the SRP and the translocon is closed. o Once the polysome binds to the receptor, the translocon opens forming a pore.  The sequence will be inserted into pore.

o The polypeptide will now continue being sequenced directly into the ER and the  signal sequence is cleaved.

o Eventually, a mature soluble secretory protein will be found in the ER lumen o This co­translational translocation requires GTP hydrolysis Don't forget about the age old question of What is co-immunoprecipitation?

∙ Post­translational Translocation  Yeast

o Some secretory proteins end up in the ER without the prior process o Some proteins, especially in yeast, are completely made in the cytosol  These proteins then react with the translocon, allowing the protein to slide  in and out of the ER lumen

 The chaperone protein (BiP) initially binds to the protein once it enters the lumen

 As more of the protein enters the cell, more chaperones are added to the  protein in order to prevent it from escaping the ER lumen

∙ Topology of Transmembrane Proteins

o Certain proteins have a special topology

 All about the orientation transmembrane protein and how it passes through the membrane

∙ Single pass or multiple pass

 The cell has to be able to put them in the membrane properly during the  synthesis

o Type I insulin receptor

 The C terminus is on the cytosolic side and the N­terminus is on the lumen side

 The initial steps mimic the soluble secretory protein up until the cleavage  of the signal sequence. 

 Once the transmembrane region is made (alpha helical region) it diffuses  out of the translocon and diffuses itself into the membrane

 The transcription of the protein will now continue freely on the cytosolic  side and NOT through the translocon

o Type II Transferrin Receptor

 There is a signal sequence but it is in the middle of the protein

 Synthesis begins on a cytosolic free ribosome

 The insertion of the protein into the lumen happens in a very specific way

∙ The protein is put into the translocon so that The N terminal is on  the cytosolic side, the protein is then cut and resynthesized in the  lumen

∙ A group of positively charged amino acid it facilitates the way  the protein was positioned

∙ The signal anchor sequence, which binds to the SRP, is the 

transmembrane region of the protein

o Type III Cytochrome P450

 This synthesis has the same orientation as type I

 Most of the protein is on the cytosolic face of the membrane

 There is no evidence of an N­terminus sequence and no evidence of SRP  involvement 

 A group of positively charged amino acids in the protein are closely  associated to the membrane on the cytosolic side

 This mechanism is a bit mysterious because its exact chain of events is not well known. 

o Type IV GLUT1 Transporter

 This is a complex synthesis

 Production of the GLUT1 transporter facilitated diffusion from one side  of the membrane to the other

 The N and C termini are on the same side, which is the cytosolic side  Synthesis begins on a cytosolic free ribosome, the N­terminus is inserted  into the translocon

∙ The synthesis continues until the first stop­transfer sequence is  reached yielding 2 transmembrane regions that diffuse into the  membrane

∙ The second set of transmembrane regions is made in the cytosol  and then inserted into the translocon then diffused into the 


o This synthesis method is used to continue the synthesis of 

the transmembrane protein until the GLUT1 molecule is 


o GPI­Anchored Proteins

 Lipid anchored proteins are also integral membrane proteins 

 GPI is covalently anchored to the C­terminus on proteins. It is initially  made as a transmembrane protein first, and then a lipid anchor protein is  added to that.

 Plasminogen activator receptor is a GPI protein

∙ It is initially synthesized like a type I, but an amino group (NH3+) is on the end of the GPI anchor

 GPI transaminase cleaves the precursor protein close to the 

transmembrane region and catalyzes the covalent attachment the lipid  anchor.

∙ ER Resident Proteins

o ER resident proteins play a role in glycosylation, disulfide bridge formation, and  folding of newly synthesized secretory proteins

o This is a group of proteins that remain in the ER because their function is in the  ER

o Binding Protein (BiP) aka. chaperone proteins, aid in folding and multimeric  assembly, prevents back sliding, and unfolded protein response.

 This ER marker helps fold proteins a they are extruded and prevents the  back sliding of proteins inserted into the ER post­translationally 

o Protein disulfide isomerases (PDI) catalyze the oxidation of free SH groups into  S­S bridges

 Facilitate the formation of disulfide bridges

o Glycosyltransferases (GTs) glycosylate proteins

∙ Unfolded Protein Response

o Unfolded proteins build up in the cell

o The cell might overexpress a protein so there is a hyperaccumulation and causes  the protein to be unable to fold

o The cell could also acquire some mutations so that when it is synthesized there is  a deformation of the 3D structure

 BiP detects this deformation and initiates an unfolded protein response ∙ BiP is bound to IRE1 an RNA nuclease that cleaves RNA

o IRE1 is a monomer.

∙ BiP releases IRE1 and attaches to unfolded proteins. When IRE1 

forms a dimer, its endonuclease activity is activated and cleaves 


∙ Hac1 is stored unspliced mRNA, but when IRE1 is activated it 

cleaves 2 exons together yielding a mature Hac1 mRNA

∙ This is then translated and a transcription factor called Hac1, 

which binds to upstream regions of genes that are involved in this 


∙ The complex then goes to the nuclease and turns on gene 

expression that is necessary for unfolded protein response. 

∙ Post­transcriptional modifications on secretory proteins by the ER

o N­linked oligosaccharide added to proteins in the ER

o N­linked is transferred to the secretory proteins from a lipid anchor to the protein  in one swoop

o It is covalently attached to asparagine N residues in the secretory proteins  Mandatory sequence: Asn­ AMINO ACID­Ser/Thr

∙ Protein Glycosylation in the RER

o Secretory protein being extruded

o Focus in the Asn residue

o The GT will transfer Asn to oligosaccharide, but before it leaves the ER and goes  to the Golgi it will be trimmed

∙ Membrane Traffic in Eukaryotic Cells

o Vesicle budding and Fusion 

 Very specific process that needs to be well controlled 

 When budded, they only pick up proteins that need to be transported  A donor compartment clips off a transport vesicle that will then fuse with  the target/ acceptor compartment 

o ER to Golgi Traffic

 Transport vesicles enter the cis face of the Golgi network and travel  through the organelle, then the molecule will exit in a secretory vesicle on  the trans face of the golgi

 There are three parts of the Golgi:

∙ Cis cisterna close to cis face

∙ Medial cisterna middle

∙ Trans cisterna close to trans face

∙ N­Linked Oligiosaccharides are processed into 2 types

o Complex

 Trimming the original N­linked glycan but also attachment of new  structures

 Addition of 2 GlcNac, 3 Gal, 3 NANA

o High Mannose

 Additional trimming of glycan that was added in the ER

∙ Functional Compartmentalization of the Golgi

o Protein Synthesis

 Cis side: phosphorylation of olgiosaccharides on lysosomal proteins  Cis cisternae: removal of Man

 Medial cisternae: removal of Man and the addition of GlcNAc

 Trans cisternae \: addition of Gal

 Trans side: addition of NANA and sorting of the protein into a lysosome  or a secretory vesicle

o Each cisterna can compartmentalize different biochemical reactions ∙ Tracking N­Glycan Processing­Biochemical 

o N­linked glycans become resistant to cleavage or sensitive to cleavage by  endoglycosylases

o Endoglycosylase H is a specific enzyme that cleaves glycoproteins  This is an important tool for trafficking studies, mainly assessing the  movement of glycoproteins through the ER and Golgi

 Analyzed using SDS­PAGE

∙ (­) is untreated and (+) is treated with endo H

∙ The (­) sample will be smaller because endo H did not cleave any 

portion of the protein off 

∙ 45 minutes is considered resistant; somewhere between 30 an 45 

minutes means it crossed the medial Golgi

 TO distinguish between secretory proteins covalently attached to high  mannose or complex N­linked Sugars

∙ High mannose is always sensitive to endo H: no matter if it is (­) or (+) endo H the protein will always be cut, which means these two 

samples will be the same size

o Endoglycosylase D

 A powerful tool to track movement of the protein throughout the Golgi giving the timing of the protein

 Between 5 and 10 minutes this protein acquired sensitive to endo D, so  that’s when the protein passed the medial Golgi

 In 10 minutes, you will have a doublet 

o Microscopy

 Expressed GFP normally on plasma membrane but are temperature  sensitive mutants

 The cells would make proteins but they would get stuck in the ER when  transferred to a lower temperature they would be released. 

∙ Secretory proteins and ER to Golgi Trafficking 

o ER resident proteins

 Selectively retrieved from the cis Golgi network

 Some proteins get caught up in the vesicle, but you need to be able to get  the proteins back

 All ER proteins have the KDEL sequence

 If one is caught in a transport vesicle, the ER resident protein can attach to the KDEL receptor in the cis golgi network, and then it is returned to the  ER

o N­Glycans are not the only sugar modification

 There are also O­linked Oligosaccharides

∙ An ­OH group bound to a Ser or Thr amino acid in the sugar

o Topology of glycoproteins

 The lumen of the ER is equivalent to the extracellular space

 If you have a transmembrane protein that is spanning the membrane,  whatever is on the lumen of the ER is on the outside of the cell

 As it moves up it ends up in the plasma membrane and vesicle fuses, the  N­linked glycan is now on the outside of the cell

o Role of glycosylation 

 Folding

∙ Contributing to the 3D structure of the proteins

 Aid in transport only rarely

∙ Targeting to lysosomes

∙ Only in one rare circumstance they participate in trafficking when  moved from the Golgi to the lysosome

 Resistant to proteases

∙ Stability

∙ Forms a coat that protects the protein from the proteases

 Protein­protein interactions

∙ Membrane traffic in Eukaryotic Cells

o Where can the proteins go after they pass through the Golgi?

 Outside the cell secreted

 Plasma membrane/ transmembrane 

 Lysosome

∙ Go through the late endosome

∙ Lysosomes are involved in the degradation of macromolecules in 

the cell

∙ All of the processes work best at an acidic pH

o The V­class pump maintains the low pH by acting as a H+ 


o Lysosomes contain acid hydrolases and other protein 

degrading molecules

∙ The cell is doubly protected

o By the membrane

o The fact that they don’t work in a pH of 7.2, also protects 

the cell if the lysosome happens to leak

∙ Lysosomes form a functional “HUB” for many cellular trafficking  pathways

o Pinocytosis, phagocytosis, autophagy

o ER Golgi

∙ How does the cell know which proteins to send to the lysosome?

o GlcNAc phosphotransferase adds the M6P only to the 

receptor on the lysosome

o GlcNAc phosphotransferase recognizes the lysosomal 

enzyme because of its signal patch

o M6P Based Traffic

 The bud comes off the trans Golgi network 

uncoated. It then fuses with a late endosome 

causing a conformational change so thet it comes 

off the receptor.

 Lysosome hydrolase loses its phosphate and it is 

moved to the lysosome, it is then recycled back to 

the trans Golgi network and used again

 If accidentally secreted, it goes to the “consecutive 

secretion” in the plasma membrane there is a M6P 

receptor and by a series of budding and fusion 

mechanism you can bring the protein back into the 

late endosome. 

∙ Plant and fungal vacuoles are versatile lysosomes

o Used for storage of waste and nutrients, degradation, and turgor pressure

o A vacuole is the lysosome in the plant cells

Cellular Endomembranes and Protein Transport Part 4  Continued

∙ Lysosomal Storage diseases

o These are enzyme deficiencies

o I-Cell disease is when a GlcNac Phosphotransferase is absent  Therefore, mannose on hydrolases not phosphorylated and  all hydrolases secreted (severe tissue destruction)

 However, liver cells (hepatocytes) in I-cell patients  

demonstrate normal distribution/targeting of hydrolases  

Therefore, different targeting mechanisms in some cells

∙ Trans Golgi Network to cell surface exocytosis and secretion o Constitutive secretion: Steady stream of vesicles from the trans  golgi membrane to the plasma membrane, usually carrying  plasma membrane proteins and soluble proteins that are  

continuously secreted

o Regulated secretion: hormones, neurotransmitters, etc. sit poised in the cytosol until a signal is transduced and received, then the  regulatory vesicle binds to the plasma membrane and released  o Anything within the vesicle is released to the outside of the cell

Cellular Endomembranes and Protein Transport Part 5 ∙ There are 3 endocytic pathways to lysosomes

o Phagocytosis: large particles are taken up by cells (engulfed from extracellular space) then fuse with lysosome

 Large particles (ex. Bacteria) are ingested

 Lower single-celled eukaryotes:  

∙ Phagocytosis = feeding

∙ used to obtain nutrients to use as building blocks

 Multicellular organisms: Phagocytosis  

∙ Defense against invading microbes/scavenging old or

damaged cells immune system!!!

o carried out be “professional phagocytes”  

(macrophages and neutrophils)

 Ex. Macrophages (defense & scavenging) and neutrophils  (defense)

 Phagocytosis is a triggered event

∙ Requires surface receptors

o Not random, the receptor must be specifically  


∙ Triggered (induced) event

o Once the ligand engages the receptor, that  

triggers the cell to engulf the particle

∙ Fc receptors binds to the constant region on the cell region A

o Endocytosis: cells take up a fluid or extremely small particle then fuse to lysosome  

 Also known as pinocytosis

 Fluid phase (& cell surface receptors) ingested  

∙ Receptor mediated endocytosis

 Usually a continuous process

 The rate depends on cell type

∙ Higher take up less  

 Ex. Macrophages take up 25% of its entire volume in fluid  phase/ hour

 Dictyostelium/Entamoeba take up > 75% or their entire  volume in fluid/hour

 Pinocytic vesicle form from clathrin-coasted pits in the  plasma membrane

∙ Clathrin-coated pits occupy 2% of total plasma  


∙ Electron dense coating made up of clathrin and other proteins, this is the site where the fluid-based  

endocytosis happens

∙ A continuous process

 Clathrin coated pts and receptor mediated endocytosis a  form of pinocytosis

∙ Receptors can end up in the endosomes by chance…  but the area is a hot spot for receptors

∙ The pit would have formed and pinched off whether  

or not the receptors were resent continuous event

o Autophagy: mechanism by which the cells get rid of organelles  that need to be degraded  

∙ Cholesterol uptake in mammalian cells

o An example of receptor mediated endocytosis

o The mechanism by which cells acquire cholesterol

 An LDL particle

o ApoB binds to the receptor

o LDL uptake  

 The late endosome has a slightly acidic pH, causes a  conformational change releasing the LDL particle,  

lysosome then degrades and releases building blocks into  the cell

∙ acidic because it pumps protons from the cytosol in,  

want it acidic so the receptor will release its ligand

 Uncoating is important for fusion to the late endosome  Where does the late endosome come from?

∙ fusion of multiple early endosomes OR a permanent  

organelle (favored)

o genetic disorder of cholesterol uptake

 LDL receptor is missing: mutation in the gene that creates  a misfolded LDL receptor causing it to be degraded

 Non-associtaion: there is a mutation where the LDL  

receptors are normal, however they cannot associate with  the clathrin coat, which means they will not be able to be  engulfed by the cell.  

 These disorders were discovered because there were a  group of patients that have a mutation that prevent the  uptake of LDL

∙ LDL builds up in their blood

∙ The fate of cell surface receptors after endocytosis

o Recycling: the receptor is returned to the membrane from which  it was engulfed

 This happens with an LDL receptor  

∙ The particle will interact with the receptors in the  

clathrin coated pit

∙ The clathrin will begin to engulf the particle into a  

coated vesicle from the plasma membrane.  

∙ The coat will then fall off, leaving an early endosome

∙ The early endosome binds with the late endosome,  

which has an acidic pH. This causes the release of  

the particle from the receptor.  

∙ The receptor is them transferred at a neutral pH to  

the plasma membrane at the surface of the cell so it  

is free and able to bind to another molecule.  

∙ The particle is then moved to a lysosome where it  

will be degraded to the building blocks of amino  

acids, cholesterol, and fatty acids

 Transferrin Cycle both the receptor and the ligand are  recycled to the original membrane  

∙ The extra cellular space has a neutral pH and the late endosome has an acidic pH

o Causing the release of iron which gives  

apotransferrin in an acidic environment

∙ The receptor will never let go of the transferrin, it  

only releases the iron molecule

∙ This cycle is responsible for delivering iron from the  

liver to all the other cells in the body

∙ The ferrotransferrin interacts with the transferrin  

receptor on the cell surface

∙ The clathrin will begin to engulf the particle into a  

coated vesicle from the plasma membrane.  

∙ The coat will then fall off, leaving an early endosome ∙ The early endosome binds with the late endosome,  

which has an acidic pH. This causes the release of  

the iron from the ferrotransferrin, creating  

apotransferrin, which then stays bound to the  


∙ The iron is then secreted into the cytosol. On the  

other hand, the apotransferrin remains bound to the  

receptor and they both are recycled to the cell  


o Degradation: the cell receptor will go into the lysosome and be  degraded

 The EGF signaling molecule, which stimulate the cells to  proliferate

∙ EGF binds to the receptor  

∙ Receptor mediated endocytosis occurs

∙ The receptor and the EGF are both degraded in the  


o Transcytosis: the cell receptor is transferred to a different  membrane from which it came from  

 Gaining antibodies from maternal milk through the  

epithelial lining to the intestine

∙ The antibody will bind to the receptor and be pulled  

into the clathrin endosome

∙ The endosome then travels from the intestinal lumen to the blood and interstitial lumen

∙ After the receptor reaches the blood and interstitial  

lumen the antibody is released and the receptor is  

transported back to the intestinal lumen through an  


∙ Trafficking pathways in eukaryotes

o Secretory ER to Golgi

o Constitutive secretion Golgi to Plasma membrane

o Regulated secretion secretory vesicle

o Endocytic movement into the cell through an early endosome ∙ Molecule Mechanisms of Vesicle Traffic

o What is the mechanism by which vesicles are formed?  There are 3 types of coated vesicles:

∙ Clathrin coated vesicles

o Clathrin and other proteins

o Movement vesicle from the Trans Golgi to the  

plasma membrane

o Clathrin coated vesicles have a tri-skeleton  structured coat that consist of 3 heavy chains  and 3 light chains.  

 This forms a cage like coat around the  


 Under a SEM it looks like a honey comb  structure

o Functions of a clathrin Coat

 Mechanical force to form vesicles

∙ The spontaneous aggregation  

event causes the mechanical force  

that results in vesicle budding  

∙ Dynamin facilitates the pinching off

of the vesicle  

o As the vesicle is forming it  

wraps itself around the  

vesicle through GTP  


o The energy it tightens and  

squeezes the vesicles off

 Capture membrane receptors  

∙ Adaptin has an affinity for the  

cytosolic tail of the receptors,  

which is why receptors tend to find  

themselves in clathrin coated pits

∙ The clathrin is the same at the  

plasma membrane and the Golgi  

membrane, but the adaptin is not  

o The adaptin in the trans gogli

network requires the  

reception of a  

phosphorylated amino acid  

on the M6P receptor

o Associated with ARF

 A monomeric GTPase that plays a role in  coat formation

 Once GDP is exchanged for GTP the ARF GTP complex becomes active and buries  

a fatty acid tail in the membrane

∙ This attracts all of the components  

of the coat to bind.  


o Coatomer-coated vesicles: large protein  complex of 7 different subunits

o Movement vesicle through the Golgi

o Has the ability to move back to the ER for the  

correction of proteins  

o These proteins function in the exact same way  

as clathrin

o Associated with ARF

 A monomeric GTPase that plays a role in  

coat formation

 Once GDP is exchanged for GTP the ARF

GTP complex becomes active and buries  

a fatty acid tail in the membrane

∙ This attracts all of the components  

of the coat to bind.  


o Coatomer-coated vesicles: large protein  

complex of 7 different subunits

o Movement of vesicles from the rough ER to the  

Cis Golgi

o These proteins function in the exact same way  

as clathrin

o Associated with Sar1

 Sar1 interacts with the receptor and  

exchanges GDP for GTP, resulting in a  

conformational change of the receptor  

which inserts a hydrophobic tail into the  

ER membrane turning it ‘on’

∙ This interaction with the membrane

will allow the COP II components to  

bind and form a coat

∙ The GTPases attract the coat  

∙ Building the coat and GTP  

hydrolysis will result in the budding

off of the vesicle

∙ Vesicle movement goes from COP II COP I Clathrin  coated vesicles

o What are the molecular signals on vesicles that cause them to  bind only to the appropriate target membrane?

o How do transport vesicles and their target organelles fuse? ∙ Monomeric-Single polypeptide  

o GNRP (guanine nucleotide releasing protein) or GEF (guanine  nucleotide exchange protein)

o Molecular switches

 Bound to GTP active

 Bound to GDP inactive

o These molecules use their own activity to hydrolyze GTP with  GTPase to get GDP, which will turn itself off

 GAP (GTPase activating protein) increases intrinsic GTPase  activity

o The C-terminus may have a prenyl or fatty acid modification ∙ SNARES and RAB GTPases play a role in vesicle traffic and fusion o Class 1: SNARES

 v-SNARES find themselves in forming vesicles  

 t-SNARES find themselves on the target vesicle

 v and t SNARES interact with each other like a lock and key model SNARE hypothesis

o Coated Vesicle Budding

 Membrane cargo receptor proteins are located within the  coated protein location, allowing soluble cargo proteins to  bind on the donor membrane and cause a cascade that will form a coated vesicle. This coated vesicle will contain a  

specific v-SNARE protein.

o Uncoated Vesicle Fusion  

 The vesicle losses its coat, forming an uncoated vesicle  that can now interact with the appropriate t-SNARE protein  on the target membrane.

 The docking of this vesicle is also assisted by Rabs

∙ Vesicle Fusion Machinery

o A lot of Rabs are involved  

o Every single vesicle trafficking event has its own Rab GTPase o Rab also plays a role, because it has its own specific receptor on  the target membrane  

 There are many different Rabs in eukaryotic cells  

 Allows for subcellular localization of some Rab GTPases

o V and T snares forma a tight and stable coil, brings the uncoated  vesicle extremely close to the target vesicle

o The exact fusion event mechanism is unknown

 Maybe the lipids in the membrane fuse together

o Disassembly of SNARE complexes

 NSF/alpha SNAP Use ATP hydrolysis energy to unravel the  snares and v-SNARE will be donated back to the donor  

membrane, but t-SNARE remains on the target membrane


∙ The cytoskeleton plays a role in the cells ability to

o Adopt a variety of shapes

o Carry out coordinated directed movements  

o Divide

o Organize its intracellular space

 Placement of organelles and movement of vesicles

∙ The cytoskeleton is a dynamic and complex network of protein  filaments that extend throughout the cell

∙ It is made up of 3 groups of proteins

o Actin filaments  

 These are helical in structure and very flexible molecules  It is a long polymer chain made up of individual actin  monomers

 There are various roles that actin can be:

∙ Microvilli and Cell cortex strengthen epithelial layers ∙ Aderens belt maintains cell to cell interaction

∙ Filopodia, Lamellipodium, stress fibers the actin is at the cell in mobile cells

o Stress fibers are on the bottom of the cell,  

where the cell contacts the surface and aids in  


∙ Phago drives the formation of the phagosomes

∙ Contractile ring helps pinch the middle of the cell to form two daughter cells

 Actin is highly abundant in the cell; a cell cannot live  without actin

 The two ends of the filament are chemically or  

biochemically different

∙ barbed end (+ end) is a fast-growing end of G actin  


∙ pointed end (- end) slow growing end (rarely see  

addition on this end)

∙ unequal growth in the two ends

o Why is there addition at + and none at – end at

physiological conditions?

 Because the critical concentration at the  

– end is much greater than the critical  

concentration at the plus end, resulting  

in a biochemical difference  

 Dynamic because addition and loss

 “treadmilling”

 Actin polymerization

∙ Nucleation this is the rate limiting step

o Monomers come together to form a seed or a  

nucleus on which the rest of the filament can  


∙ Elongation

o Addition much more easily of monomers

 Mainly on + end, sometimes on –

∙ Steady state

o Loss of monomer and gain of monomers are  equal

 no change in length

o Critical concentration of actin monomers  

 Toxins that alter polymerization and depolymerization  dynamics of actin by perturbing the steady state ∙ Cytochalain: Bind to the + end of actin, preventing  any more monomers joining the + end but allows the loss on the – end, depolymerization

∙ Latrunculin: binds to G actin monomers, preventing  them from being incorporated into the + end  

polymer. Still allows – to loss monomers,  


∙ Phallodin: binds to the side filaments preventing  monomers from coming off – end and prevents  addition to + end, essentially freezing the polymer  Actin binding proteins

∙ Polymerization

o Motility happens rapidly

 Extension of the front end

 Adhesion; putting down a new adhesion

 Translocation moves the body forward

 De-adhesion is the depolymerization at  

the back, allowing it to bring it back  

toward the center

 Profilin and Thymosin B4

 How does the cell change direction?

∙ relying on the protein complex Arp  

2 3.

∙ this is responsible for allowing the  

actin to branch and grow in a  

different direction  

∙ NPF binds to G actin subunit

∙ Interacts with Arp2/3 complex

∙ Interacts with an actin filament

∙ Creates a new positive end on the  

old filament, allowing a new branch

to grow

 Cell gets a signal to change direction  

causing Arp 2/3 to bind to the side of the  


 Listeria Monocytogenes use actin  

polymerization to move through cells ad  

jump from cell to cell

∙ Bacteria can hijack the actin  

polymerization machinery

∙ Produces a protein that can  

dissolve the phagosome  

membrane, which means it was  

never delivered to the lysosome for

destruction. Free floating in the cell

∙ So strong it will create microspikes  

∙ Circumvents the immune system  

∙ Act A mimics the function of NPF,  

creating a lot of + ends near the  

back of the bacterium, which will  

push it forward

 The role of actin in endocytosis

∙ Proteins on the surface which can attract the NPFs

∙ when that happens, you get NPF bound to actin,  

attracting Arp2/3 yielding active polymerization to  

form a vesicle

∙ This vesicle can then be moved in a similar fashion  

as the cells, through the use of actin

∙ Phagocytosis is a triggered event, causing the  

membrane to engulf through the recruitment of actin

growth around the phagosome

 The role of Actin in the cell shape

∙ Filament is a cross linking agent  

∙ All the same cell (platelets) relying on actin  

∙ Filamin can cross link actin, changing its shape of the cytoskeleton and in turn the shape of the cell

o Intermediate filaments

 Intermediate in size and flexibility

 Sole function is to provide structural integrity to tissues  Form a protective coat around the nucleus

o Microtubules

 Very rigid polymers of tubulin monomers that form a hollow tube

 They are often found emanating from a microtubule  

organizing center, not found freely floating in the cell

o Intermediate filaments and microtubules have a similar pattern  because intermediate filaments are commonly found on the side  of microtubule

∙ Actin based motor proteins­Myosins

o Myosins

 Protein that uses the energy from ATP hydrolysis to move along a certain  filament.

 Myosin = motor protein in actin

 17 different classes of myosin – large protein family

 Most abundant is myosin II

  myosin II – 2 heave chains and 4 light chains

 “large globular head domain” active site for ATP binding and hydrolysis,  actin binding site also here. 

 Neck domain or hinge region – plays role in motility of myosin along  actin – how far and how fast is dictated by the size of the neck. 

o Important Members of the Myosin Family

 Myosin I

 Myosin II (muscle myosin)

 Myosin V

∙ All have head, tail, neck region.

∙ #light chains (bind calcium that regulates activity) varies among  the different members

∙ Vesicle trafficking – bind to tail region of myosin and head region  will walk along actin and that will help move the vesicles along.

∙ Myosin I – responsible for organizing actin in the cell. 

o ATP hydrolysis by myosin head domains and motor activity

 How myosin moves along the filament

  1) head is bound to 1 actin monomer (green). Tail region bound to  something else 

 2) Myosin bind ATP and you get a change of conform in the neck region  such that the myosin head will come off actin

 ATP hydrolyzed – cause conformational change and myosin steps down  and will bind another g actin monomer towards the plus end of the  filament.

 All mysosins except myosin 6 will move towards the plus end.

o Myosin II Model

 Prediction of Model:  Distance traveled in one step, and thus velocity  should be directly proportional to neck length 

∙ Conformational changes in neck facilitate all this movement.  Step size or speed at which a myosin can move is dictated by the length of  its neck. 

 Recombinant myosins with different lengths of neck above – bigger neck  takes bigger steps

o Myosin V Steps

 Both globular heads moving along actin (model before only showed 1 but  there are 2)

 Two Proposed Mechanisms:

∙ Alternating Leading Heads (walking)

∙ Permanent leading head (shuffling)

 Testing the Hypothesis

∙ Position along an actin filament

∙ ­added fluores mysosiins and measured the position – they could 

predict the step size.

∙ All cases the step size was 72 nm and suggests the alternating 

hypothesis was correct.

∙ Function of Myosins

o Function of Myosin I, II, and V

 Myosin I helps to organize actin relative to the cell membrane – when it  moves to the plus end it moves entire filament.

 Myosin II – muscle contraction

 Myosin V – tail region attached to vesicle, moves towards plus end  dragging the vesicle along (from donor to target)

o Myosin II molecules can aggregate to form “Thick Filaments”

 II – only myosin that can aggregate to form these thick filaments (dozens  of myosin tubes).

o General Structure of Skeletal Muscle

 Myosin II referred to as “muscle myosin”

 Muscle cell long cell called myofiber – zoom in and you will find that it is  loaded of bundles of myosin II and actin (myofibrils)

 Myofibrils made up of units called sarcomere (z disk to z disk)

o Molecular Structure of Sarcomere

 Actin filaments attached by z disk – z disk is a large group of proteins   Plus ends of actin filaments are interacting/buried in z disks.

 In between actin filaments are myosin thick filaments 

 A band defines where the myosin thick filaments

 H defines where there is no actin

 I goes from a to a (not important)

o Actin and Myosin Binding Proteins that Promote Stability“Capping Proteins”  Required when actin structures must remain relatively stable or static (i.e. muscle cells)

 CapZ (actin binding)­binds to + end of actin filaments

 Tropomodulin (actin binding)­binds to – end of actin filaments

 Nebulin (actin binding)­binds to the side of actin filaments

 Titin (myosin binding)­binds to myosin and Z disk proteins

∙ All above proteins stabilize sarcomere structure

o Actin Capping proteins add stability to the Sarcomere Structure  Cap Z helps with Z disk interaction with actin

 Tropo ­ Do not want deploy or poly – want constant length important for  muscle contraction

o Titin (Myosin binding protein) and Nebulin (Actin binding protein) maintain  organization of sarcomere

 Titin maintains structural stability

o Sliding Filament Model

 Add ATP and Ca, all heads move towards plus end.

 Two z disks come closer to each other, which causes sarcomere to contract (imagine all sarcomeres contracting at the same time which facilitates  muscle contraction). 

o Role of Myosin II in Non Muscle Cells

 Dividing – 

∙ contractile ring – made up of actin and myosin II

∙ myosin II facilitates pinching of contractile ring that causes the  cells to divide. Treated with anti­myosin, will not divide, have a  multi nucleated cell

 Epithelial –

∙ adhesion belt – near the apical side. Made up of actin and myosin  II

∙ helps epithelial cells to bind to their neighbors. During 

development, these cells will pinch off. Like a drawstring pouch to cause epithelium to pinch off. 

 Fibroblast­

∙ Large groups of actin near the surface – actin and myosin II

∙ Myosin II plays a role in structural integrity.

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