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