Biol110-10-Lecture 10-Vesicles

Biol110-10-Lecture 10-Vesicles - Vesicle-mediated protein...

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Unformatted text preview: Vesicle-mediated protein transport within cells: Mechanisms of vesicle budding, targeting and fusion Fundamental questions in membrane transport•  How to deform fluid membranes into vesicles? •  How to package proteins into vesicles? •  How vesicles find their target organelle? •  How do their membranes fuse? •  How is the vesicle machinery recycled? targeting budding fusion Vesicle-mediated trafficking between organelles of the secretory pathway- Retrograde direction Anterograde direction exomer An assay that monitors vesicle budding & differential effect of inhibitors- Assay measures vesicle budding as: release of “protease protected” ERglycosylated proalpha factor (a secreted protein) from perforated cells. Same Sec protein requirements as in vivo…therefore, in vitro reaction likely represents what happens in vivo Use of inhibitors to block different stages of the ERGolgi transport pathway Inhibitors stabilize transient intermediates (e.g. transport vesicles). Inhibition of the GTPase Rab1/Ypt1p led to the purification of ‘free-floating’ ER-derived transport vesicles Purification of ER-derived transport vesiclesVesicles accumulate in vitro in the presence of an inhibitor (Ypt1p-mutant) that blocks vesicle targeting to the Golgi. Vesicles were purified based on their size and density, and their content of radiolabeled core-glycosylated pro-alpha-factor Protein composition of ER-derived vesicles (without their coat)Transmission electron micrographs of purified vesicles that have shed their coats… why? Vesicles + then sediment What kind of protein are the ERVs? These ER-Vesicle (ERV) proteins turned out to be integral membrane receptors for cargos Vesicle budding from ER microsomes using purified coat proteins- Coated vesicles accumulate in the presence of a nonhydrolysable analog of GTP The most abundant proteins in coated-vesicle preparations are the coat proteins. Vesicle-bud nucleation is driven by the GTPase Sar1p and its binding to membraneThe site for vesicle assembly on the ER membrane is marked by the guanine exchange factor (GEF) Sec12, which activates (and recruits) the Sar1 GTPase to the membrane Phospholipid bilayer Sar1p then recruits coat proteins, which in turn recruit cargo Sec12p Completing the assembly of COPII vesicle coats and capture of cargosThe Sec13p-Sec31p subcomplex polymerizes onto Sec23pSec24p and crosslinks the pre-budding complexes Sec24p contains at least three distinct sites for capturing cargo, but all subunits of the coat can presumably bind cargos. Only the membrane proximal COPs can potentially bind (and sort) phospholipids The concave face of the Sec23-Sec24 complex explains how the membrane is curved…the BAR domains. Cytosolic Sar1pGDP is converted to membrane bound Sar1pGTP by the transmembrane protein Sec12p. Sar1pGTP recruits the Sec23p-Sec24p subcomplex by binding to Sec23p, forming the “pre-budding complex”. Transmembrane cargo proteins gather at the assembling coat by binding to Sec24p. The Sec13p-Sec31p subcomplex polymerizes onto Sec23p-Sec24p and crosslinks the pre-budding complexes. Cargo proteins are further concentrated. The depictions of Sar1p, Sec23p, and Sec24p are surface representations from the crystal structures of these proteins. The Sec13p-Sec31p complex is represented as an elongated, five-globular domain structure based on electron microscopy. Sec16p and Sed4p also participate in the assembly of COPII, but are not shown here because their roles are less well understood. Protein (and lipids) need to be sorted for incorporation into vesicles during coat assemblyMost proteins are sorted prior to incorporation into vesicles, but abundant proteins may enter by bulk flow Most ER resident proteins are retained by a tether However, most ER resident proteins enter vesicles by mistake at some point and need to be retrieved • Lipid sorting • Membrane scission Identity of signals and adapters? Vesicle scission is accomplished by the polymerization force of coat assembly, or the Sar1 hydrophobic tail? The issue of COPII vs. clathrin… Biochemical evidence of protein sorting during vesicle budding (prior to scission) Use vesicle budding assay in perforated yeast Vesicle release Some proteins (e.g. BiP/Kar2) are retained in the ER Signals that direct membrane proteins (cargo) to anterograde ER-derived transport vesicles- It is recognized by the COPII subunit Sec24p Anterograde sorting signals that direct secretory proteins to ER-derived vesicles- Oops! Sorting signals are also used for retrieving proteins from the Golgi membranes- Signals that direct proteins to retrograde transport vesicles are recognized by receptorsKDEL retrieval signal and its receptor: Confers “retention” of lumenal proteins in the ER, by retrieving them if they leave the ER by mistake, rather than holding them in the ER as chaperones do Some ER lumenal proteins share the amino acid sequence KDEL at their C-terminus. If KDEL is removed, the protein is secreted If KDEL is added to a secreted protein, that protein is retained in the ER… Golgi Dilysine signal: Confers retrieval of transmembrane proteins to the ER via binding to COPI alpha-subunit ER ** Demonstration using the HDEL vs KDEL signals of different yeast… Uncoating of transport vesicles occurs following vesicle scission from the ER- Uncoating is blocked by nonhydrolyzable analogs of GTP Uncoating is triggered via GTP hydrolysis by Sar1p. One of the COPII subunits (Sec23p) is a Sar1p GTPase activating protein (GAP). Uncoated vesicles display the cytoplasmic domains of membrane proteins (the vSNARE’s) that will target the vesicles to cis-Golgi membrane. Vesicles that cannot shed their coats, will not fuse with membranes. Uncoated ER-derived transport vesicles fuse to form a tubulo-vesicular network (also termed ERGIC) Uncoated vesicles and target membranes display proteins involved in vesicle docking and fusionvSNAREs impart identity to vesicles tSNAREs impart identity to the target membrane v-t SNARE complexes lock the vesicle unto its target and are very stable (slow off rate) Tetherins ‘reach out’ and grab an approaching vesicle; they have long coil-coiled domains Another example of Rab function: Rab1/Ypt1-GTP removes a deactivating ‘cap’ protein (Sly1) from a tSNARE (Sed5)) Crystallization of vSNARE-tSNARE complexes and the current SNAREpin zipper hypothesis- Zippering-up of vSNAREs and tSNAREs The SNARE complex is a bundle of four helices Parallel orientation of v and t SNAREs during SNARE-pin assembly bring membranes together; an antiparallel attachment would be unproductive Membrane fusion via v-t SNARE zippering and its intermediates- Unstable hemifusion intermediate NSF and SNAPs function in the disassembly of v-t SNARE pairs that form during membrane fusion, v-SNAREs are then recycled After membrane fusion, the ATPase NSF and its cofactor a-SNAP utilize energy from ATP hydrolysis to unfold the SNARE-complex, returning SNAREs to the initial monomeric state. The SNARE cycle. A trans-SNARE complex assembles when a monomeric v-SNARE on the vesicle binds to an oligomeric t-SNARE on the target membrane, forming a stable four-helix bundle that promotes fusion. The result is a cis-SNARE complex in the fused membrane. SNAP binds to this complex and recruits NSF, which hydrolyzes ATP to dissociate the complex. Unpaired v-SNAREs can then be packaged again into vesicles by interacting with coat proteins Vesicular traffic at the synapse Vesicle docking with the major players in place vSNARE: Synaptobrevin tSNAREs: Syntaxin and Snap-25 Synaptotagmin: A Ca2+ sensor Ca2+ channel Exocytosis The SNARE complex is the target of several neurotoxinsSites for tetanus toxin (TeNT) and botulinum toxin (BoNT) cleavage Syntaxin (tSNARE) Synaptobrevin/VAMP2 (vSNARE) SNAP25 (tSNARE) These toxins functions as highlyspecialized proteases that cleave synaptic SNAREs causing paralysis of victims by blocking vesicle fusion with plasma membrane in synapses, thereby interrupting nerve impulse transmissions. Most current view of vesicle targeting and fusion mechanisms Membrane fusion on the exocytic and endocytic pathways, in five steps. (a) The first association of membranes, termed ‘tethering’, requires a prenylanchored Rab-family GTPase and tethering proteins termed ‘effectors’124, which bind to the Rab in its activated, GTP-bound state. Proteins mediating tethering have been studied in the Golgi stacks125,126 and other systems. (b) Rab-regulated enrichment of fusion proteins and lipids in a microdomain. Rabs, their multi-functional effector complexes, and lipids with defined roles in fusion (such as sterols (not shown) or phosphoinositides or diacylglycerol (red polar head groups)) assemble into a microdomain, the site of subsequent steps in the fusion pathway. In some systems, Rab effectors can include guanine nucleotide exchange factors, which activate Rabs; lipid kinases, which synthesize phosphoinositides; and SM proteins, which bind SNAREs. It remains unclear in most instances whether Rab effectors must remain bound to the Rab to be activated for these functions, or whether concentration of these several protein and lipid factors in the fusion microdomain suffices. In some systems, such as the yeast vacuole, one multisubunit complex fulfills tethering, guanine nucleotide exchange, SM protein, and lipid-binding functions. (c) Assembly of trans-SNARE complexes127 with additional regulatory proteins. These include SM proteins48 and can include proteins or domains that bind to Ca2+, to lipids or to SNAREs. These complexes may encircle the site of future fusion44. Lipids with small head groups and negative membrane curvature, which promote hemifusion, are enriched at the cytoplasmic surface of the fusion microdomain (red head groups). (d) Hemifusion, formed by fusion of the halves of the lipid bilayer of each membrane that face the cytoplasm. Arrows indicate the direction of subsequent lipid movement to complete the fusion process. Lipids with positive curvature due to large head groups (shown here as blue head groups) may become enriched at this stage for invasion of the hemifused structure (arrows). (e) Completion of fusion, with mixing of lipid bilayers, membrane proteins and luminal compartments but retention of the barrier between cytoplasm and organellar lumen. This process converts trans-SNARE complexes to postfusion cis-SNARE complexes; it is unclear whether cis-SNARE complexes can arise by any other route. SNAP (Sec17p) may displace other SNAREbound proteins and prepare the cis-SNARE complex for ATP-dependent disassembly by NSF (Sec18p). A comparison between COPII membrane coats and nuclear pore membrane coats COPII membrane coat Sar1 Sec23-24 complex cytoplasm Sec31-13 complex Nuclear pore membrane coat cytoplasm Nup82 Gle1 Nup59/53 Rtn1/Yop1 nuclear envelope Mlp1 Nup188/192 Nup170/157 Nup84 complex Ndc1 Pom34 Pom152 Nup1 Nup60 Sec12 cargo peripheral ER Nic96 nucleus “Piecing together nuclear pore complex assembly during interphase” (2009) Rexach, M; J Cell Biol185; pg 377-9 ...
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This note was uploaded on 01/17/2011 for the course BIOL 110 taught by Professor Rexach during the Fall '10 term at UCSC.

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