Vesicular Traffic, Secretion & Endocytosis PDF

Vesicular Traffic, Secretion & Endocytosis  Secretory Pathway The secretory pathway is so named because it was initially studied in dedicated secretory cells that produce and secrete large quantities of proteins such as: Insulin Digestive enzymes  Fates of Proteins Synthesize...

Vesicular Traffic, Secretion & Endocytosis  Secretory Pathway The secretory pathway is so named because it was initially studied in dedicated secretory cells that produce and secrete large quantities of proteins such as: Insulin Digestive enzymes  Fates of Proteins Synthesized on Bound Ribosomes Proteins made that follow the secretory pathways but are not meant to stay in the ER or the Golgi have the following fates: – Regulated Secretion – Constitutive Secretion – Directed to the Lysosome – Insertion into the Plasma Membrane Proteins delivered to the plasma membrane include 1. cell-surface receptors 2. Transporters for nutrient uptake 3. Ion channels Secreted proteins include: Digestive enzymes Peptide hormones Serum proteins collagen  Principle of secretion Common principle: transport of membrane & soluble proteins from one membrane-bound compartment to another is mediated by transport vesicles The vesicles collect “cargo” proteins in buds arising from the membrane of one compartment & deliver the “cargo” to the next compartment by fusing with the membrane of that compartment at least 3 different types of vesicles are required for transport Secretory & Endocytic Pathways that Sort Proteins Newly made proteins incorporated into the ER lumen or membrane Packaged into anterograde transport vesicles – forward-moving Vesicles fuse with each other to form cis-Golgi cisterna – flattened membrane-bound compartment Some proteins are retrieved back to the ER via retrograde transport vesicles – backward-moving  Secretory & Endocytic Pathways that Sort Proteins Cisternal progression – New cis-Golgi cisterna with “cargo” move cis to medial to trans position – Retrograde transport of enzymes & Golgi-resident proteins needed at each position constantly retrieved Reach the trans-Golgi network (TGN) = major branch point where cargo is loaded into 1 of 3 vesicles: – Exocytosis: continuous secretion Secretory vesicles: regulated secretion; stored inside cell until a signal comes for exocytosis – Transport vesicle to lysosome via a late endosome (proteins can also enter this way via endocytosis) Vesicular Stomatitis Virus (VSV) membrane glycoprotein fused to Green Fluorescent Protein (GFP): Visualized protein transport  Molecular mechanism of Vesicle budding and fusion Vesicles move from parent/ donor organelle to daughter organelle Donor organelle contain integral membrane proteins v-SNAREs that join with their cognate t-SNAREs in target membrane  3 Types of Coated Vesicles Each have different protein coat Each formed by reversible polymerization of distinct protein subunits 1. COPII: transports in the anterograde direction from the RER to the Golgi. 2. COPI: transports in the retrograde direction b/w Golgi cisternae and from the cis-Golgi back to the RER. 3. Clathrin coated vesicles: transports between the plasma membrane or trans-Golgi to late endosomes proteins of the coated vesicles that transport proteins from the trans-Golgi to the plasma membrane for regulated or constitutive secretion are unknown.  Assembly of Protein Coats Conserved set of GTPase switch proteins control the assembly of different vesicle coats – All vesicles contain a GTP-binding protein that regulate assembly. COPI and Clathrin vesicles contain ARF (ADP-ribosylation factor) COPII contains Sar1 ARF and Sar1 belong to the GTPase superfamily  COPII Vesicle Synthesis Sar1-GDP is recruited by Sec12 on the parent membrane Sec12 catalyzes the reaction that exchanges GDP for GTP binding of GTP causes a conformational change in Sar1 conformational change exposes a hydrophobic N-terminal region that tethers the Sar1-GTP complex to the membrane complex drives polymerization of COPII proteins & coat formation  COPII Vesicle Synthesis The coat forms and the vesicle buds from the parent membrane.  COPII Vesicle Synthesis Sar1 GTPase activity w/ help of some COPII sub- units hydrolyze GTP which triggers protein coat dissociation Cargo protein dissociated coat Cargo receptor exposes integral proteins v-SNARE protein called v-SNAREs  Targeting Cargo Proteins vesicles select cargo proteins by binding sorting sequences present on cargo proteins – cargo targeting sequences make specific molecular contacts w/ coat proteins – vesicle coat selects cargo molecules by directly binding specific sorting signals on cytosolic portion of membrane cargo proteins – polymerized coat is an affinity matrix that clusters selected membrane cargo proteins into forming vesicle buds  Docking of Vesicles to Target Membranes Requires another set of GTP- binding proteins known as Rab proteins (Rab GTPases) Cytosolic Rab-GDP is converted to Rab-GTP, causing a conformational change that enables the Rab-GTP complex to anchor to the vesicle membrane. Rab-GTP complex binds to a Rab effector on the target membrane.  Docking of Vesicles to Target Membranes cytosolic Rab – GDP Rab - GTP Rab effector on the Target membrane Docking of Vesicles to Target Membranes This allows for the fusion of the membrane & formation of SNARE complexes – VAMP = v-SNARE on the vesicular membrane – (VAMP- vesicle associated membrane protein) – Syntaxin & SNAP-25 = t-snare on the target membrane Multiple non-covalent interactions stabilize this structure Docking of Vesicles to Target Membranes After fusion, the Rab-GTP is hydrolyzed and released. SNARE complex dissociates: needs ATP hydrolysis and other cytosolic proteins. NSF – N-ethylmaleimide sensitive factor (NEM- sensitive factor) NSF associates with the SNARE complex with the aid of the α-SNAP The NSF then hydrolysis the ATP, releasing sufficient energy to dissociate the SNARE complex  Vesicle-mediated Protein Trafficking between the ER & cis-Golgi Anterograde Retrograde transport transport Mediated by Mediated by COPI COPII vesicles vesicles Moves proteins Recycles to the Golgi membrane bilayer & some proteins Shortly after the vesicle release, the coat is shed exposing proteins required for fusion  Role of KDEL & KDEL Receptor Retrieval of ER-resident luminal proteins from the Golgi. KDEL –Lys-Asp-Glu- Leu sequence. Usually as the C-terminal This retrieval system prevents depletion of ER luminal proteins such as those needed for proper folding of newly made secretory proteins  Golgi Apparatus composed of membrane-bound sacs known as cisternae – b/w 5-8 usually present, but