Lysosome Nucleus ER Plasma Membrane Mitochondria Golgi

A variety of coat complexes participate in vesicle formation

COP-II Coat Components Coat Assembly COP-II Coat Components 1) GDP-Sar1p binds to Sec12p 2) GTP/GDP exchange 3) GTP-Sar1p anchors to membrane “Sec” refers to secretory mutants in yeast develop by Randy Scheckman.

Monomeric G-proteins Regulate COP-II Coat Assembly GEF = guanine nucleotide exchange factor GAP = GTPase activating protein Sar1 = ras-like G-protein Sec12 = Sar1-specific GEF Sec23 = Sar1-specific GAP

COP-II Coat Components Coat Assembly COP-II Coat Components 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 “Sec” refers to secretory mutants in yeast.

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/t-SNARE 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? COP-I also in Golgi BFA: originally ascribed to both anterograde and retrograde transport targeting dictated by SNARES BFA: loss of Golgi dilation of ER Golgi markers in ER rapidly reversible coats prevent premature fusion

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