Mitochondria
PlasmaMembrane
NucleusLysosome
ER
Golgi
A variety of coat complexes participate in vesicle formation
Coat Locations G-proteinCOP-II ER ERGIC Sar1
COP-I(coatomer)
ERGIC ER; Golgi stacks;endocytic compartments
ARF1
clathrin +adaptors
TGN; cell surface (receptor-mediated endocytosis)
dynamin;ARF1
retromers (?) endosome Golgi
caveolin (?) cell surface
COP-II Coat Components
1) GDP-Sar1p binds to Sec12p2) GTP/GDP exchange 3) GTP-Sar1p anchors to
membrane
Protein SizeSar1p 21 kDaSec12p 43 kDaSec23-complex 400 kDa
Sec23p 85 kDaSec24p 105 kDa
Sec13-complex 700 kDaSec13p 34 kDaSec31p 150 kDa
“Sec” refers to secretory mutants in yeast develop by Randy Scheckman.
Coat Assembly
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
1) GDP-Sar1p binds to Sec12p2) GTP/GDP exchange 3) GTP-Sar1p anchors to
membrane4) Sec23p-Sec24p complex
binds to GTP-Sar1p5) Sec13p-Sec31p complex
binds next
Protein SizeSar1p 21 kDaSec12p 43 kDaSec23-complex 400 kDa
Sec23p 85 kDaSec24p 105 kDa
Sec13-complex 700 kDaSec13p 34 kDaSec31p 150 kDa
“Sec” refers to secretory mutants in yeast.
Coat Assembly
Vesicle Formation• driven by coat assembly• cargo is concentrated
• SNAREs implicated• p24 family?
• ER resident proteins are excluded (Sec61) and/or retrieved (BiP, SNARE)
• 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
Transport Vesicles Uncoat and Dock with Destination Compartment
• 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
SNAREs Determine Specificity of Vesicle Docking
• 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)
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
• 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
• 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)
COP I Components and Assembly
• COP-I also in Golgi• 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
Golgi and beyond?
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