5 A variety of coat complexes participate in vesicle formation
6 COP-II Coat Components Coat AssemblyCOP-II Coat Components1) GDP-Sar1p binds to Sec12p2) GTP/GDP exchange3) GTP-Sar1p anchors to membrane“Sec” refers to secretory mutants in yeast develop by Randy Scheckman.
8 COP-II Coat Components Coat AssemblyCOP-II Coat Components1) GDP-Sar1p binds to Sec12p2) GTP/GDP exchange3) GTP-Sar1p anchors to membrane4) Sec23p-Sec24p complex binds to GTP-Sar1p5) Sec13p-Sec31p complex binds next“Sec” refers to secretory mutants in yeast.
9 Vesicle Formation driven by coat assembly cargo is concentrated SNAREs implicatedp24 family?ER resident proteins are excluded (Sec61) and/or retrieved (BiP, SNARE)
10 Transport Vesicles Uncoat and Dock with Destination Compartment GTP-Sar1p converted to GDP-Sar1p following vesicle releaseactivated by Sec23pGDP-Sar1p dissociatespromotes coat disassemblyuncoating exposes SNAREs(SNAP receptor)mediate docking and fusion2 types: vesicle and targetv-SNARE binds t-SNARE
11 SNAREs Determine Specificity of Vesicle Docking t-SNARE (=syntaxin family)8 members in yeastall in different compartment (except 2 on plasma membrane)each binds specific v-SNARE (eg., Sed5p/Sft1p)rab checks fit between SNAREsmonomeric G-proteinGTPase ‘locks’ complex
12 Membrane Fusion Machinery SNAP binds to v/t-SNARE complexNSF only binds to SNARE-SNAP complexactivation of NSF associated ATPasefusion mechanism not knownNSF = NEM-Sensitive Fusion Protein (Sec18)Sec18 required at all steps in secretory and and endocytic pathwaysNSF binding requires cytosolic factorSNAP (Soluble NSF Attachment Protein)
13 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?
14 COP-I vesicles responsible for retrograde transport KDEL signal (eg., BiP)analogous to COP-II
15 COP I Components and Assembly ARF1 (ras-like G-protein) + 7 COPs (coat proteins)coatomer (, , ', , , , and )GTP-ARF1 binds to membraneanchored by myristic acidARF1 receptor unknownbrefeldin A (BFA) inhibits GEFmembrane bound ARF1 recruits coatomerbudding and vesicle formationGTP hydrolysis leads to dissociation of coatomerdocking and fusion (SNARE, SNAP, and NSF)
16 Golgi and beyond? COP-I also in Golgi BFA: originally ascribed to both anterograde and retrograde transporttargeting dictated by SNARESBFA:loss of Golgidilation of ERGolgi markers in ERrapidly reversiblecoats prevent premature fusion
17 Problems with Vesicular Transport Model requires additional t-SNARES or mechanisms for COPI bidirectionalityno evidence for anterograde movement of COPI vesiclesresident Golgi proteins demonstrate gradient-like distribution across cisternaelarge structures like algal scales or procollogen precursorsA recent rebirth of cisternae maturation model