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Lecture 5

Lecture 5 - Vesicular Traffic of Proteins, Golgi Apparatus

6 Pages

Course Code
Biology 2382B
Sashko Damjanovski

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LECTURE 5: VESICULAR TRAFFIC OF PROTEINS, GOLGI APPARATUS Overview of Secretory and Endocytic Pathways of Protein Sorting Secretory pathway: synthesis of proteins bearing an ER signal sequence is completed on the rough ER (1) and newly made polypeptide chains are inserted into the ER membrane or crossed into the lumen. Some proteins remain within the ER. Remainder are packaged into transport vesicles (2) that bud from the ER and fuse to form the new cis-Golgi cisternae. Missorted ER-resident proteins and vesicle membrane proteins that need to be reused are retrieved to the ER by vesicles (3) that bud from the cis-Golgi and fuse with the ER. Each cis-Golgi cisterna, with its protein content, physically moves from the cis to the trans face of the Golgi complex (4) by nonvesicular process called cisternal maturation. Retrograde transport vesicles (5) move Golgi-resident proteins to the proper Golgi compartment. In all cells, certain soluble proteins move to the cell surface in transport vesicles (6) and are secreted continuously (constitutive secretion). In certain cell types, some soluble proteins are stored in secretory vesicles (7) and are released only after the cell receives an appropriate neural or hormonal signal (regulated secretion). Lysosome-destined membrane and soluble proteins, which are transported in vesicles that bud from the trans-Golgi (8), first move to the late endosome and then to the lysosome. Endocytic pathway: membrane and soluble extracellular proteins taken up in vesicles that bud from the plasma membrane (9) also can move to the lysosome via the endosome. How do we get proteins into the Golgi complex? • The secretory pathway and mechanisms of vesicular traffic allow proteins to be secreted from the cells or delivered to the plasma membrane and the lysosome o This pathway carried both soluble and membrane proteins from the ER to final destination at the cell surface or in the lysosome • In contras to the secretory pathway which allows proteins to be targeted to the cell surface, the endocytic pathway is used to take up substances from the cell surface and move them into the interior of the cell o Used to ingest certain nutrients that are too large to be transported across the plasma membrane by one of the transport mechanisms o Can also be used to remove receptor proteins from the cell surface as a way to down-regulate their activity • Anterograde transport – movement of proteins towards Golgi • Retrograde transport – movement of proteins from Golgi to ER Golgi Complex • Consists of flattened, disklike, cisternae with no ribosomes • Vesicles at cisterna tips fuse or pinch off • Three types of cisternae (cis, medial, and trans) and two flanked networks of tubules: CGN faces RER and TGN is opposite to RER • Processing and sorting of proteins (secreted, membrane, lysosomal) Transport Vesicles: Budding and Fusion • Coat proteins promote budding of vesicles • Snare proteins promote fusion of vesicles with target membranes • COPII vesicles → RER to Golgi (anterograde transport) • COPI vesicles → cis-Golgi to RER (retrograde transport) • Clathrin vesicles → TGN/PM to late endosomes • Two types of cargo protein: o Free floating within the lumen of the ER o Membrane-associated protein that has part facing the lumen, and another facing cytoplasm • Membrane-cargo receptor protein o Passes through membrane, also has cytosolic component o Can bind certain soluble cargo proteins and help make sure they get incorporated in newly forming vesicle • GTP-binding proteins o Help promote association of coat proteins with ER membranes (COPII) o Help facilitate binding in combination with cargo receptor proteins a) Budding is initiated by recruitment of a small GTP-binding protein to a patch of donor membrane. Complexes of coat proteins in the cytosol then bind to the cytosolic domain of membrane cargo proteins, some of which also act as receptors that bind soluble proteins in the lumen, thereby recruiting luminal cargo proteins into the budding vesicle. b) After being released and shedding its coat, a vesicle fuses with its target membrane in a process that involves interaction of cognation SNARE proteins. • Transport of membrane and soluble proteins from one membrane-bound compartment to another is mediated by transport vesicles that collect “cargo” proteins in buds arising from the membrane of one compartment and then deliver these cargo proteins to the next compartment by fusing with the membrane of that compartment • As transport vesicles bud from one membrane and fuse with the next, the same face of the membrane remains oriented toward the cytosol • Goal: get proteins within ER or associated with membrane of ER from the ER to the Golgi apparatus o Mediated by vesicles • Cargo proteins are soluble • Binding to membrane cargo receptor will trigger an event (shift in protein) – make the cytoplasmic protein recognizable to coat protein; GTP-binding protein will bind GTP o These will promote budding, physical change (“putting the coat on”) • SNARE proteins (v-snare – vesicle snare proteins; t-snare – target snare proteins) twist around each other and fuse the membranes between vesicle and target membrane – specific joining of v-SNAREs in vesicle membrane with t-SNAREs in target membrane to which the vesicle is docked brings the membranes into close apposition, allowing the two bilayers to fuse • All transport vesicles use v-SNAREs and t-SNAREs to bud and fuse, regardless of target organelle • Coat proteins o COPII coat proteins form at RER membrane – anterograde o COP I coat proteins help vesicles form at Golgi – retrograde GTP binding proteins control assembly and disassembly of COPII coat proteins • Membrane associated GTP binding proteins promote association of COPII coat proteins on ER membrane • Once COPII vesicles are released from donor membrane, hydrolysis of GTP occurs which triggers disassemble of COPII coat proteins • GTP has to be bound to the protein – help promote association of COPII coat proteins on ER membrane • Hydrolysis of GTP to GDP triggers dissociation event – disassembly of GTP-binding protein and COPII coat proteins RER to cis-Golgi Transport • COPII vesicles mediate anterograde transport o GTP binding proteins control (1) assembly and disassembly of COPII coat proteins and (2) docking vesicles to target membrane. • Certain cargo membrane proteins use DXE (Asp-X-Glu) sorting signal recognized by COPII proteins. • ATP hydrolysis is required for dissociation of SNARE complexes. • COPI vesicles mediate retrograde transport between Golgi cisternae and ER. • Certain integral ER membrane proteins are specifically recruited into COPII vesicles for transport to the Golgi • The cytosolic segments of many of these proteins contain a diacidic sorting signal (key residues areAsp-X-Glu or DXE) • DXE is important for coat protein to bind to cytoplasmic component of membrane receptor • DXE is important for dissociation of COPII proteins and is essential for selective export of certain membrane proteins from the ER • SNARE proteins twist and promote diffusion • ATP hydrolysis is needed to disassemble SNARE proteins • SNARE proteins are required for fusion of vesicles (COPII vesicle and its fusion to protein membrane) • COPI proteins will bind to cis-membrane, promote budding; formation of COPI vesicle will be directed back to ER and fuse with SNARE protein • COPII is important for going up, COPI is important for going down Cystic Fibrosis • Recessive genetic disease • Characterized by abnormal transport of chloride and sodium across epithelium, leading to thick, viscous secretions • Affects lung, liver, pancreas and intestine • Caused by a mutation in the gene for the protein cystic fibrosis transmembrane conductance regulator (CFTR). • F508 is most common mutation in CFTR • Affects ability of CFTR to bind COPII coat proteins • CFTR has a diacidic (DXE) sorting signal • In order to contract CF, both alleles have to be mutated (autosomal recessive) • CFTR transports ions (especially Na and Cl) across lung epithelium cells and other cells • Not being able to transport Na and Cl affects osmosis (movement of water) and not being able to move water results in the outsides of cells becoming dry • Secreted mucous becomes thick and viscous – can decrease lung cell capacity/breathing facility, and also easily harvests bacteria • Major problem: transporter can’t get to
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