Exam 2 Study Guide
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This 41 page Study Guide was uploaded by Luke Holden on Saturday October 8, 2016. The Study Guide belongs to BIOL 4610 at Clemson University taught by Susan Chapman in Fall 2016. Since its upload, it has received 196 views.
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Date Created: 10/08/16
Exam 2 Study Guide for Dr . Chapman Contains: Week 4 Lecture Notes Week 5 Lecture Notes Week 6 Lecture Notes Tables Comparing and Contrasting Cytoskeleton All Process required to know Helpful figures and diagrams Helpful Quizlets Links Helpful Video links Hope this Helps!!!!!! BiP- Binding Proteins (Member of the Hsp 70) Heat Shock Protiens (Hsp70) These are an abundant chaperon proteins in the ER lumen that assist with the folding of cells More specifically the are called BiP- Binding Proteins These guys bind to the hydrophobic regions on the nacent protein that is being cotranslated into the ER lumen o This helps bring the protein in as well as keep it from aggregating with other proteins o It is very important because thousands of proteins are being pushed into the ER and if they all stuck together then they all would be unable to work. Protein Disulfide Isomerase (PDI) Important Things to know about PDI Disuldife bonds are found on cysteine molecules This molecule helps with structure and folding of a protein Soluble Sulfur is crucial You want your bond to look like this: How PDI Works o IT IS ESSENTIAL THAT YOU KNOW REDOX REACTIONS LIKE THE BACK OF YOUR HAND!! o “THIS WILL BE AN EXAM QUESTION” o OIL RIG: 1. Oxidation involves loss (or having loss) of a proton 2. Reduction involves gain (or having gain) of a proton 3. all proteins want to be stable They would rather die (in no way are they living but just metaphorically speaking) than be unstable Unfolded protein Response o ERAD (ER Associated Degradation) Proteasomes come and degrade protein sugar molecules are crucial for this recognition Protein Routing is controlled by many factors: many proteins synthesized in the ER are glycoproteins which are tissue specific insital glycosylation occurs as soon as the protein enters the golgi complex helps modify protein KDEL (Lys-Asp-Glu-Leu) o These are for proteins whose final destination is the ER o The Golgi complex has a receptor protein that binds the KDEL sequence and delivers the protein back to the ER ON HER SLIDESHOW FOR “PRESENTAITON SLIDE SET 4- ENDOMEMBRANES II” STOP HERE!!! YOU DON’T HAVE TO KNOW ANY OF THEE MATERIAL BEYOND THIS POINT OF THIS POWERPOINT BEGIN WITH SLIDE 30 ON “Slide Set 5- protein targeting and sorting” (we will cover mitochondrion but just to keeps thing consistent with the ER we are starting here). Glycosyltransferases Glycosylation- the addition of carbohydrate side chains to proteins Glycoproteins are made here and come with a modified oligosaccharide side chain N-Linked O-Linked Nitrogen-Linked Oxygen-Linked Addition of and oligosaccharide to Addition of and oligosaccharide Serine Asparginine or threonine N-Linked: Refer to slide 33 This is the big picture on where the glycosylated protein goes On the Next page we will talk about how to make this We want to make this: two units of N-acetylglucosamine, nine mannose units, and three glucose units Mitochondrion and Protein Targeting Facts: o you get your mito from your mother o 57 known genes in the mito o mito obviouslt need more than this o gets extra genes from the nucleus o so its kind of like a we need you but you need us relationship we need the mito power they need the nucleus DNA o mito encoded : o mito- encoded are transcribed in the mito (THIS IS NOT A MISTAKE! PLEASE KNOW THAT THIS IS A SPECIAL KIND OF TRANSCRIPTION BECAUSE IT TAKES PLACE IN THE MITO o mito- encoded translated on its OWN ribosomes in the intracellular ribosomes o other proteins are also transcribed and translated in the mito o Nuclear Encoded o The nuclear encoded proteins are just like the old stuff you know where it is transcribed in the nucleus and then translated in the cytosol o then it is imported into the mitochondrion o Importing proteins into the mito o Requires 4 protein/ protein complexes Import Receptor reads mitochondrial targeting signal delivers to translocon Translocon of Outer Membrane (TOMs) associates with import receptor delivers protein to second translocon Translocon of Inner Membrane (TIMs) aligned with TOM40 Matrix Hsc70 (acts like BiP) aids in net translocation o Targeting sequences for the mito (starting to notice a pattern here?) this sequence is hydrophobic, basic, and hydroxylated residues and is cleaved once it enters the mito N-terminus located 20-25 AA long forms and amphipathic helix o Most of the proteins are folded in the cytosol o Hsc 70 proteins come and bind to the newly synthesized protein and hydrolyze ATP to keep the protein linear (like a shoe string) o Bringing in the protein to the matrix on the next page! o Applying proteins to the mitochondrial membrane is a different story: o The orientation HAS TO BE CORRECT!!!! o 3 path ways to a mitochondrial membrane o Chart on next page Target Example Steps Picture Sequence Path Hydrophob Cytochrom Import A ic Stop- e oxidase receptor transfer subunit TOM 40 sequences TIM Complex Signal Cleavege Released by TIM into IM Path Internal STP TOM40 B sequences synthase TIM recognized subunit 9 Signal by Oxa 1 Cleavage Released into matrix Oxa1 inserts into IM Path Mulitple ADP/ADP Import C sequences antiporter receptor- recognized (TOM70/TOM by TOM 70 20) and TIM 22 TOM 40 TIM(22/54) released by TIM into inner membrane and facilitated by TIM9 and 10 Golgi Apparatus o ER and glogi are almost continuous o Structure and function of golgi: Don’t have to know all the steps just important fx include: Sugar molecules are added and removed Terminal sugar is modified (this is like the golgi tool box) it can craft the sugar and make it whatever it wants it to be. o Transport Vesicles This supplies thee movement of solutes through the golgi o 2 Models o The Stationary Cisternae Model In this model, each cisterna in the Golgi stack is a stable structure Transport of materials from one cisterna to another is mediated by shuttle vesicles These bud off from one cisterna and fuse with the next cisterna in a cis-to-trans sequence o The Cisternal Maturation model In this model, each cisterna in the Golgi stack is a stable structure Transport of materials from one cisterna to another is mediated by shuttle vesicles These bud off from one cisterna and fuse with the next cisterna in a cis-to-trans sequence Retrograde movement (Cop1): Backwards Movement of protein back to ER after final modification This allows the cell to balance the flow of lipids toward the plasma membrane It also ensures a supply of materials for forming new vesicles Proteins whose final destination is the ER have a KDEL sequence (Lys-Asp-Glu-Leu) or related sequence The Golgi complex has a receptor protein that binds the KDEL sequence and delivers the protein back to the ER Anterograde movement (Cop3): Forwards Movement of material toward the plasma membrane is called anterograde transport As a secretory granule fuses with the plasma membrane and discharges its contents (exocytosis), a bit of membrane from the ER becomes part of the plasma membrane This flow of lipids toward the plasma membrane must be balanced Cytoskeleton Microtubules Microfilaments Intermediate Filaments Size 25nm 7nm 8-12 nm Structure MTs are straight, G-actin monomers The fundamental hollow cylinders of can polymerize subunits of IF varied length that reversibly into proteins are consist of (usually filaments with a dimers 13) longitudinal lag phase and arrays of polymers elongation phase, IF proteins are called similar to tubulin fibrous rather than protofilaments assembly globular The basic subunit F-actin filaments Each has a of a protofilament are composed of homologous is a heterodimer of two linear strands central rod like tubulin, one α- of polymerized G- domain of 310 to tubulin and one actin wound into a 318 amino acids in β-tubulin helix length These bind noncovalently to All the actin Flanking the form an monomers in the central helical filament have the domain are N- and αβ-heterodimer, same orientation C-terminal which does not domains that differ normally G-Actin and F-Actin greatly among IF dissociate proteins GTP and ATP 1 IF= IMPORTANT: IF 4 protofibril YOU ARE GOING α- 72 nm long for one 16 protofiliments α OR β-β THEN turn 32 dimers YOU CAN CHANGE Ropelike 64 polymers THE GTP. BUT IF YOU GO α-β, THEN Monomer YOU CAN’T threshold (Kind of CAHNGE GTP!!!!!! Like MT) Subunits α and β Subunits 1 Strand of G-Actin 1 IF= Each one is 3-D I monomers= G 4 protofibril identical by 40% actin strand 16 protofiliments matched AA wise 2Stands of Gactin 32 dimers monomers wound 64 polymers Each has an N- together is an F- terminal GTP- Actin polymer binding domain, a central domain to which colchicine can bind, and a C- terminal domain that interacts with MAPs (microtubule- associated proteins) All the dimers in the MT are oriented the same way Because of dimer orientation, protofilaments have an inherent polarity The two ends (called the plus end and the minus end) differ both chemically and structurally Most organisms have several closely related genes for slight variants of α- and β-tubulin, referred to as tubulin isoforms Function Organization of Organization of primarily intracellular intracellular mechanical, organelles and organelles and transport of transport of intermediate vesicles (motor vesicles (motor filaments are less proteins) proteins) dynamic than actin filaments or Beating of cilia Intracellular microtubules. and flagella Motility (e.g. Shigella) Intermediate Nerve cell, red filaments blood cell, and Cellular Stability commonly work in flagellar structure (Stress Fibers) tandem with microtubules, Alignment and Cellular Motility providing strength separation of (Crawling) and support for chromosomes the fragile tubulin during mitosis and Muscle Contraction structures. meiosis muscle-specific actins (α-actins) Microtubules (Highways of the cell) (Largest) Functions Organization of intracellular organelles and transport of vesicles (motor proteins) Beating of cilia and flagella Nerve cell, red blood cell, and flagellar structure Alignment and separation of chromosomes during mitosis and meiosis Characteristics Unstable Form o mitotic apparatus o short lived o assembles quickly and then dissembles just as quick Stable Form o long lived o remains polymerized for a long time o An example is cilia on egg: moves it along the oviduct; if you don’t have this you will be sterile 2 Types of MT o Cytoplasmic (Unstable) Functions: Maintaining axons Formation of mitotic and meiotic spindles Maintaining or altering cell shape Placement and movement of vesicles o Axonemal microtubules (Stable) Functions in: Cilia Flagella Basal bodies to which cilia and flagella attach The axoneme, the central shaft of a cilium or flagellum, is a highly ordered bundle of MTs Structure (How they are made) MTs are straight, hollow cylinders of varied length that consist of (usually 13) longitudinal arrays of polymers called protofilaments The basic subunit of a protofilament is a heterodimer of tubulin, one α-tubulin and one β-tubulin These bind noncovalently to form an αβ-heterodimer, which does not normally dissociate IMPORTANT: IF YOU ARE GOING α-α OR β-β THEN YOU CAN CHANGE THE GTP. BUT IF YOU GO α-β, THEN YOU CAN’T CAHNGE GTP!!!!!! Subunit Structure o Each one is 3-D I identical by 40% matched AA wise o Each has an N-terminal GTP-binding domain, a central domain to which colchicine can bind, and a C-terminal domain that interacts with MAPs (microtubule-associated proteins) o All the dimers in the MT are oriented the same way o Because of dimer orientation, protofilaments have an inherent polarity o The two ends (called the plus end and the minus end) differ both chemically and structurally o Most organisms have several closely related genes for slight variants of α- and β-tubulin, referred to as tubulin isoforms Don’t have to know isoforms How they form o MTs form by the reversible polymerization of tubulin dimers in the presence of GTP and Mg 2+ o Dimers aggregate into oligomers, which serve as “seeds” from which new MTs grow o This process is called nucleation; the addition of more subunits at either end is called elongation o THIS IS IMPORTANT THE AMOUNT OF DIMERS NEEDED FOR AN MT TO GROW NEEDS A THREASHOLD VALUE!!!!!!!!! MT FORMATION REQUIRES A SPECIFIC NUMBER OF MOLECULES TO START THE PLUS END THRESHOLD VALUE IS LOWER THAN THE MINUS END. THUS THE MT WILL GROW IN THIS DIRECTION!!!!!! THIS IS BECAUSE THERE IS A GREATER GTP BINDING CAPACITY!!! Facts you need to know about formation o Free tubulin exists as a/b dimer o b(GTP) dimer will add onto (+) end o Addition of next dimer will trigger GTP hydrolysis in b-subunit o Spontaneous nucleation is rare in vivo; need MTOCs When the mass of MTs reaches a point where the amount of free tubulin is diminished, the assembly is balanced by disassembly; this is known as the plateau phase The elongation phase is much faster MT formation is slow at first because the process of nucleation is slow; this period is known as the lag phase Some Definitions you need to know Centrosome- is two centrioles Critical Concentration- The tubulin concentration at which MT assembly is exactly balanced by disassembly o MTs grow when the tubulin concentration exceeds the critical concentration and vice versa Treadmilling addition of subunits at the plus end, and removal from the minus end If the free tubulin concentration is above the critical concentration for the plus end but below that of the minus end, treadmilling will occur Microtubules Can Form as Singlets, Doublets, or Triplets Cytoplasmic MTs are simple tubes, or singlet MTs, with 13 protofilaments (this is like what we have been talking about Some axonemal MTs form doublet or triplet MTs (Cilia, Flagella) Doublets and triplets contain one 13-protofilament tubule (the A tubule) and one or two additional incomplete rings (B and C tubules) of 10 or 11 protofilaments (basal bodies, centrioles) Instability of MT GTP and its relation to this whole process o Each tubulin heterodimer binds two GTP molecules; α-tubulin binds one, and β-tubulin binds a second o The GTP bound to the β-subunit is hydrolyzed to GDP after the heterodimer is added to the MT o GTP is needed to promote heterodimer interactions and addition to MTs, but its hydrolysis is not required for MT assembly Dynamic Instability Model (Catastrophe) o one population of MTs grows by polymerization at the plus ends, whereas another population shrinks by depolymerization o Growing MTs have GTP at the plus ends, and shrinking MTs have GDP o The GTP cap at the plus end prevents subunit removal o If GTP-tubulin is high, it is added to an MT quickly, creating a large GTP- tubulin cap o If the concentration falls, the rate of tubulin addition decreases o At a sufficiently low GTP-tubulin, the rate of GTP hydrolysis exceeds the rate of subunit addition, and the cap shrinks o When it pulls apart, it pulls apart like a banana. Proteofiliment by proteofiliment. Catastrophe and Rescue o If the GTP cap disappears altogether, the MT becomes unstable, and loss of GDP-bound subunits is favored o If there are no dimers then catastrophe will occur because any dimmer present cannot be added by the time the β- GTP is hydrolyzed. o Individual MTs can go through periods of growth and shrinkage; a switch from growth to shrinkage is called microtubule catastrophe o A sudden switch back to growth phase is called microtubule rescue Centriole Structure Centriole walls are formed by nine pairs of triplet microtubules They are oriented at right angles to each other They are involved in basal body formation for cilia and flagella Cells without centrioles have poorly organized mitotic spindles Quizlets for Microtubules: https://quizlet.com/158184431/drugs-that-affect-the-cytoskeleton-exam-2-flash- cards/ https://quizlet.com/158187935/proteins-involved-in-microtubles-flash-cards/ Videos for Microtubules https://www.youtube.com/watch?v=-qV__tYb4c4 γ- TubulinRC (ring complex) Centrosomes have large ring-shaped protein complexes in them; these contain γ-tubulin γ-tubulin is found only in centrosomes γ-tubulin ring complexes (γ-TuRCs) nucleate the assembly of new MTs away from the centrosome Loss of γ-TuRCs prevents a cell from nucleating MTs Adds to the (-) end No γ- Tubulin= Death Microtubules Originate from Microtubule-Organizing Centers within the Cell MTs originate from a microtubule-organizing center (MTOC) Many cells have an MTOC called a centrosome near the nucleus In animal cells, the centrosome is associated with two centrioles surrounded by pericentriolar material (-) end is in the MTOC MTs grow outward from the MTOC with a fixed polarity—the minus ends are anchored in the MTOC Because of this, dynamic growth and shrinkage of MTs occurs at the plus ends, near the cell periphery Gamma-tubulin ring complex plays key role in nucleation MTOC in Dividing Cells Each MTOC has a limited number of nucleation and anchorage sites MT-nucleating activity can be modulated during processes such as mitosis Centrosomes associated with spindle poles in mitotic cells have highest MT- nucleating activity while the spindle is forming Some more Stability Proteins Tip Proteins o MTs can be stabilized by proteins that “capture” and protect the growing plus ends o These are +–TIP proteins (+–end tubulin interacting proteins) o Proteins such as EB1 (end-binding protein 1) decrease the likelihood that MTs will undergo catastrophic subunit loss o Microfiliments Characteristics Smallest (7nm) They are best known for their role in muscle contraction They are involved in cell migration, amoeboid movement, and cytoplasmic streaming Development and maintenance of cell shape (via microfilaments just beneath the plasma membrane at the cell cortex) Structural core of microvilli actinmyocin Microvilli How They are formed G-actin monomers can polymerize reversibly into filaments with a lag phase and elongation phase, similar to tubulin assembly F-actin filaments are composed of two linear strands of polymerized G- actin wound into a helix All the actin monomers in the filament have the same orientation G-Actin and F-Actin GTP and ATP 72 nm long for one turn Ropelike Monomer threshold (Kind of Like MT) Need that Critical Concentration to begin formation Polarity of MF Myosin subfragment 1 (S1) can be incubated with microfilaments (MFs) S1 fragments bind and decorate the actin MFs in a distinctive arrowhead pattern Because of this pattern, the plus end of an MF is called the barbed end, and the minus end is called the pointed end The polarity of MFs is reflected in more rapid addition or loss of G-actin at the plus end than the minus end After the G-actin monomers assemble onto a microfilament, the ATP bound to them is slowly hydrolyzed So, the growing MF ends have ATP-actin, whereas most of the MF is composed of ADP-actin Therefore, as you increase the age of MF then you increase the amount of ADP in the MF Again, adds to the plus end more readily We will talk about these more in depth Treadmilling is also found in these guys (you don’t have to know the math!) Structures Commonly Found Structures: Leading Edge (Filopodium) (needed for crawling) o (+) End towards the outside of the cell (adds monomers) o (-) End towards the inside of the cell o microfilaments form highly oriented, polarized cables with the plus ends toward the tip of the protrusion o o Lamellipodium (needed for crawling) o Branched Network of actin o There is order o Gel o There is no order o Stress Fiber Microvilli Actin bundles in microvilli are the best-studied examples of ordered actin structures Microvilli are prominent features of intestinal mucosal cells; they increase the surface area of the cells The core of a microvillus consists of a tight bundle of microfilaments with the ends pointed toward the tip The MFs are connected to the plasma membrane by crosslinks made of myosin I and calmodulin The MFs in the bundle are tightly bound together by crosslinking proteins fimbrin and villin The Terminal Web o At the base of the microvillus, the MF bundle extends into a network of filaments called the terminal web o The filaments of the terminal web are composed mainly of myosin II and spectrin, which connect the MFs to each other, to proteins in the membrane, and perhaps also to intermediate filaments Proteins that link Actin to membranes MFs are connected to the plasma membrane and exert force on it during cell movement or cytokinesis This (indirect) connection to the membrane requires one or more linking proteins Examples of such proteins include band 4.1, ezrin, radixin, moesin, spectrin, and ankyrin Proteins That Promote Actin Branching and Growth Actin can also form a dendritic (treelike) network A complex of actin-related proteins, the Arp2/3 complex, nucleates new branches on the sides of filaments o causes severe immune deficiency if mutated Arp2/3 branching is activated by a family of proteins that includes WASP (Wiskott-Aldrich syndrome protein) and WAVE/Scar Long Actin Filaments For some cell functions, long actin filaments are needed In this case, actin polymerization is regulated independently of the Arp2/3 complex, through proteins called formins Formins move along the end of the growing filament as they promote polymerization Rho • Both plasma membrane lipids and regulatory proteins affect the formation, stability, and breakdown of MFs • The cytoskeleton of cells exposed to certain growth factors can undergo a dramatic change • Many signals exert their effects by acting on phospholipids, but they also act through a family of monomeric G proteins called Rho GTPases • 3 Classes • Rho pathway-results in formation of stress fibers • Rac- activation results in extension of lamellipodia • Cdc42- activation results in the formation of filopodia Regulation of Rho GTPases Rho GTPases are stimulated by guanine-nucleotide exchange factors (GEFs) through the exchange of bound GDP for GTP GTPase activating proteins (GAPs) inactivate Rho GTPases by causing them to hydrolyze their bound GTPs to GDP Guanine-nucleotide dissociation inhibitors (GDIs) sequester inactive Rho GTPases in the cytosol Intermediate Filaments Characteristics Intermediate filaments (IFs) are not found in cytosol of plant cells but are abundant in many animal cells An abundant intermediate filament (IF) is keratin, an important component of structures that grow from skin in animals IFs are the most stable and least soluble components of the cytoskeleton They likely support the entire cytoskeleton Rope like as well Very Different Chemically inactive Tensile Strength Problems with holding tissue together Can be elastic Intermediate Filaments Confer Mechanical Strength on Tissues Intermediate filaments are thought to play a tension-bearing role In neurons, IFs are dynamically transported and remodeled IFs are less susceptible to chemical attack than are MTs and microfilaments The Cytoskeleton Is a Mechanically Integrated Structure Microtubules resist bending when a cell is compressed Microfilaments serve as contractile elements that generate tension Intermediate filaments are elastic and can withstand tensile forces Intermediate Filament Proteins Are Tissue Specific IFs differ greatly in amino acid composition from tissue to tissue They are grouped into six classes: o Class I: acidic keratins o Class II: basic or neutral keratins o Proteins of classes I and II make up the keratins found in epithelial surfaces covering the body and lining its cavities o Class III: includes vimentin (connective tissue), desmin (muscle cells), and glial fibrillary acidic protein (GFAP) (glial cells) o Class IV: the neurofilament (NF) proteins found in neurofilaments of nerve cells o Class V: includes the nuclear lamins A, B, and C that form a network along the inner surface of the nuclear membrane o Class VI: nestin, the substance that makes up the neurofilaments in nerve cells of embryos o Animal cells can be distinguished based on the types of IF proteins they contain—a technique known as intermediate filament typing Intermediate Filaments Assemble from Fibrous Subunits The fundamental subunits of IF proteins are dimers IF proteins are fibrous rather than globular Each has a homologous central rod like domain of 310 to 318 amino acids in length Flanking the central helical domain are N- and C-terminal domains that differ greatly among IF proteins One Model of Intermediate Filament Assembly The basic structural unit consists of two IF polypeptides intertwined into a coiled-coil The two polypeptides are aligned in parallel Two such dimers align laterally to form a tetrameric protofilament Protofilaments overlap to build up a filamentous structure about eight protofilaments thick Integration of Cytoskeletal Elements Spectraplakins are linker proteins that connect intermediate filaments, microfilaments, and microtubules One, called plectin, is found at sites where intermediate filaments connect to MFs and MTs Quizlets for MF https://quizlet.com/158188492/proteins-involved-with-microfiliments-flash-cards/ https://quizlet.com/158184431/drugs-that-affect-the-cytoskeleton-exam-2-flash- cards/ Videos https://www.khanacademy.org/test-prep/mcat/cells/cytoskeleton/v/microfilaments- and-intermediate-filaments-2 Cellular Movement and Motility Microfilaments Actin-Based Motility o Crawling Motility o Actin and myosin have been discovered in nearly all eukaryotic cells o They are known to play important roles in nonmuscle motility The Process o Cell Migration via Lamellipodia Involves Cycles of Protrusion, Attachment, Translocation, and Detachment Protrusion Many nonmuscle cells are capable of crawling over a substrate using lamellipodia and/or filopodia Cell crawling involves distinct events: extension of a protrusion, attachment to substrate, and generation of tension, which pulls the cell forward Retrograde Flow: Retrograde flow results from actin assembly at the growing tip of the protrusion and rearward translocation of filaments toward the base Arp2/3-dependent branching drives actin polymerization, particularly in lamellipodia Microtubules are also involved in the process Attachment Attachment, or adhesion, of the cell to the substrate is necessary for cell crawling New sites of attachment are formed at the front of the cell, and contacts at the rear must be broken Attachment sites are complex structures involving a number of proteins Integrins: o Integrins on the outside of cells attach to extracellular matrix proteins o Inside the cell, integrins are connected to actin filaments via linker proteins o The integrin-dependent attachments are called focal adhesions Translocation and Detachment Contraction at the rear of the cell squeezes the cell body forward and releases the attachments at the rear Contraction, due to actin-myosin interactions, is under control of Rho, which activates nonmuscle myosin II at the rear of the cell For movement to occur, new attachments must be balanced by loss of old ones o Motility vs Contractility Motility Movement of a cell or organism through the environment Movement of the environment past or through a cell Movement of components in the cell Motility occurs at the tissue, cellular, and subcellular levels Intracellular components move; for example, microtubules of the mitotic spindle play a role in the separation of chromosomes during cell division To generate movement, microtubules (MTs) and microfilaments (MFs) provide a scaffold for motor proteins that produce motion at the molecular level 2 Types of Systems from Eukaryotes o Microtubule-based motility Examples: fast axonal transport in neurons; the sliding of MTs in cilia and flagella o Microfiliment-Based motility Muscle Contraction Contractility used to describe shortening of muscle cells, is a specialized form of motility o All about the engine that drives them along They couple ATP hydrolysis to changes in shape and attachment of the motor protein They undergo cycles of ATP hydrolysis, ADP release, and acquisition of new ATP They have common structural features They can move along a cytoskeletal filament for significant distances Microtubules Motors o Kinesins (mostly + end) anterograde Kinesins consist of two dimerized heavy chains and two light chains The heavy chains contain globular domains that attach to microtubules, a coiled-coil stalk, a lever-like neck that connects the two, and a tail The light chains are associated with the tail Kinesins are classified into families based on their amino acid sequence Some form homodimers; others, heterodimers Kinesin-14 is minus-end directed; kinesin-5 is bidirectional The kinesin-13 family, the catastrophins, aid depolymerization of MTs. Movement along MT Kinesin movement looks like “walking,” with the two globular head domains taking turns as the front foot Each kinesin molecule exhibits processivity It can move long distances along an MT before detaching from it by releasing bound ADP and acquiring a new ATP, so that the cycle repeats 1 ATP=1 step=8nm o Dynein (mostly – end) retrograde (The drunken sailor walk) So think of Dyneins as the drunken sailor pulling a trailer. The Dynactin is the bed of the trailer and acts as Velcro for the vesicle to sit on. o o Movement inside cells o MTs provide a rigid set of tracks for transport of a variety of organelles and vesicles o Traffic toward the minus ends of MTs is considered “inbound” (dyneins); toward the plus end is “outbound” (kinesins) o Microtubule-associated motor proteins—kinesins and dyneins—walk along the MTs and provide the force needed for movement Axonal Transport Proteins produced in the cell body are transported to the nerve ending in a process called fast axonal transport This involves packaging of these proteins into vesicles for transport Organelles can also be observed moving along filaments through axoplasm (cytoplasm of axons) at rates of about 2 μm/sec ( So, for a vesicle to travel down the axon from the spinal cord to the foot, it would take about 5 days! (This is going really fast even through it does not seem like it. Proteins responsible o Kinesin I is involved in ATP-dependent transport toward the plus ends (away from the centrosome), called anterograde axonal transport o Cytoplasmic dynein moves particles (cargo) in the opposite direction, called retrograde axonal transport The Process Page BiP Binding to Protiens to bring in Proteins o When the protein is pushed into the cell, ATP is HYDROLYZED and BiP clamps down on to the protein o When BiP wants to release, it simply re-phosphorylates converting ADP to ATP and the protein is released This classifies it as an ATPase o As a way of quality control (to make sure the protein folded correctly) if the hydrophobic binding region is on the inside of the cell then the protein is correct o If the hydrophobic region is on the outside then BiP rebinds and it tries again PDI Forming a sulfide bond Reduced protein comes along to meet up with an oxidized PDI PDI is reduced (gets the proteins electrons) as the sulfur on the Cys gets oxidized This positive charge (electrophile) is attached by the next sulfur (nucleophile) on the protein The second sulfer is then oxidized giving the proton to PDI Now you have a DI sulfide bond PDI is then oxidized by ERO1 (which ERO 1 is reduced) now PDI can return and help for another disulfide bond. Steps for Fixing a Disulfide Bond We don’t know how this happens but we thing it is because PDI can enter the hole created by the misfolded protein and a correctly folded protein does not leave a hole PDI now starts REDUCED it comes and donates (oxidized) electrons to the misbonded disulfide thus the reaction is reversed Then the process can resume as normal Glycosylaiton Glycosylation begins as dolichol phosphate, an oligosaccharide carrier, is inserted into the ER membrane GlcNAc and mannose groups are then added to the phosphate group The growing core oligosaccharide is translocated to the ER lumen by a flippase Once inside the lumen, more mannose and glucose are added The completed core oligosaccharide is transferred from dolichol to the asparagine residue of the recipient protein The core oligosaccharide attached to the protein is trimmed and modified Once it has reached the end of the line it is transferred all together (en massae) to the protein From there is under goes quality to control check before being exported to the golgi: Quality Control in the ER The glucoses are taken off one by one until it reaches the end. If all is well a mannose is removed and it is exported to the golgi If not all is well then a glucose will reattach to the mannose and it will stay in ER for a long time If not fixed than it is destroyed (coming up next) So: 321 are we good? Yes Mannose GO (2Glc and 8 Mann) 321 are we good?--> No Glucose and sit (2Glc 9 mann I Glucose) To remember this process just think pf Dr. Chapman saying Chop, Chop, Chop. Unfolded Protein Response Remember, these are the proteins that get stuck in the ER for to long that cant be fixed So, this is like a recycling process in which BiP is brought back because BiP is so important. It does this by: 1. Binding to the BiP proteins in the ER Lumen which were original attached to the 2 monomers Ire1. 2. The release of BiP from the monomer casue the two Ire1 monomers to form a dimer which activates ints endonuclease activity 3. Next, the pre spliced Hac1 mRNA comes and the intron in spliced out by the Ire1 dimer 4. The mature mRNA is then translated and the resulting Hac1 protein returns to the nucleus where it catalyzes the transcription of BiP ( a protein folding catalyst) Protein Folding calnexin (CNX) and calreticulin (CRT) (they are both lectins- sugar molecule attachers) These bind to monoglucosylated glycoproteins and assist with protein folding This occurs in a complex that includes thiol reductase know as ERp57 The complex is removed and the final glucose is removed by glucosidease II Steps o glucosyl transferase, UGGT, (UDP-glucose:glycoprotein glucotransferase), binds to improperly folded proteins o It adds back a single glucose unit, making the protein a substrate for CNX/CRT binding o Once proper conformation is achieved, UGGT no longer binds the new glycoprotein, which moves on to the Golgi Bringing in the Mitochondrial Membrane Proteins Step 1: The import receptor binds the protein and prepares it for entry Step 2: TOM and TIM line up creating a hole directly to the matrix the space (don’t have to know the names of tom and tim but know that TOM 40 is the main translocon of the outer membrane) note that if a protein is just going to the IMS then it skips TIM! Step 3 Upon entry of the matrix the targeting sequence is then cleaved off by the matrix processing protease and new Hsc 70 (matrix) proteins bind to keep that protein straight Step 4: The protein is finally translated HIGHLY BASIC IN THE MATRIX THIS FURTHER INTIATES PROTEIN FOLDING
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