Transcript Powerpoint
Lysosome Nucleus ER Plasma Membrane Golgi Mitochondria A variety of coat complexes participate in vesicle formation Coat Locations G-protein COP-II ER ERGIC Sar1 COP-I (coatomer) ERGIC ER; Golgi stacks; ARF1 endocytic compartments clathrin + adaptors TGN; cell surface (receptor- dynamin; mediated endocytosis) ARF1 retromers (?) endosome Golgi caveolin (?) cell surface COP-II Coat Components Protein Sar1p Sec12p Sec23-complex Sec23p Sec24p Sec13-complex Sec13p Sec31p Size 21 kDa 43 kDa 400 kDa 85 kDa 105 kDa 700 kDa 34 kDa 150 kDa “Sec” refers to secretory mutants in yeast develop by Randy Scheckman. Coat Assembly 1) GDP-Sar1p binds to Sec12p 2) GTP/GDP exchange 3) GTP-Sar1p anchors to membrane Monomeric G-proteins Regulate COP-II Coat Assembly • Sar1 = ras-like G-protein • Sec12 = Sar1-specific GEF • Sec23 = Sar1-specific GAP GEF = guanine nucleotide exchange factor GAP = GTPase activating protein COP-II Coat Components Protein Sar1p Sec12p Sec23-complex Sec23p Sec24p Sec13-complex Sec13p Sec31p Size 21 kDa 43 kDa 400 kDa 85 kDa 105 kDa 700 kDa 34 kDa 150 kDa “Sec” refers to secretory mutants in yeast. Coat Assembly 1) GDP-Sar1p binds to Sec12p 2) GTP/GDP exchange 3) GTP-Sar1p anchors to membrane 4) Sec23p-Sec24p complex binds to GTP-Sar1p 5) Sec13p-Sec31p complex binds next Vesicle Formation • driven by coat assembly • cargo is concentrated • SNAREs implicated • p24 family? • ER resident proteins are excluded (Sec61) and/or retrieved (BiP, SNARE) Transport Vesicles Uncoat and Dock with Destination Compartment • GTP-Sar1p converted to GDP-Sar1p following vesicle release • activated by Sec23p • GDP-Sar1p dissociates • promotes coat disassembly • uncoating exposes SNAREs • (SNAP receptor) • mediate docking and fusion • 2 types: vesicle and target • v-SNARE binds t-SNARE SNAREs Determine Specificity of Vesicle Docking • t-SNARE (=syntaxin family) • 8 members in yeast • all in different compartment (except 2 on plasma membrane) • each binds specific v-SNARE (eg., Sed5p/Sft1p) • rab checks fit between SNAREs • monomeric G-protein • GTPase ‘locks’ complex Membrane Fusion Machinery • SNAP binds to v/tSNARE complex • NSF only binds to SNARE-SNAP complex • activation of NSF associated ATPase • fusion mechanism not known • NSF = NEM-Sensitive Fusion Protein (Sec18) • Sec18 required at all steps in secretory and and endocytic pathways • NSF binding requires cytosolic factor • SNAP (Soluble NSF Attachment Protein) • vesicle formation at ER driven by COPII • COPII vesicles fuse to form ERGIC • (ER-Golgi Intermediate Compartment) • aka VTC (Vesicular-Tubular Clusters) • return of ER components? • COP-I vesicles responsible for retrograde transport • KDEL signal (eg., BiP) • analogous to COP-II COP I Components and Assembly • ARF1 (ras-like G-protein) + 7 COPs (coat proteins) • coatomer (, , ', , , , and ) • GTP-ARF1 binds to membrane • anchored by myristic acid • ARF1 receptor unknown • brefeldin A (BFA) inhibits GEF • membrane bound ARF1 recruits coatomer • budding and vesicle formation • GTP hydrolysis leads to dissociation of coatomer • docking and fusion (SNARE, SNAP, and NSF) Golgi and beyond? BFA: • loss of Golgi • dilation of ER • Golgi markers in ER • rapidly reversible • coats prevent premature fusion • COP-I also in Golgi • originally ascribed to both anterograde and retrograde transport • targeting dictated by SNARES Problems with Vesicular Transport Model • requires additional t-SNARES or mechanisms for COPI bidirectionality • no evidence for anterograde movement of COPI vesicles • resident Golgi proteins demonstrate gradient-like distribution across cisternae • large structures like algal scales or procollogen precursors A recent rebirth of cisternae maturation model