1. Lysosome
Nucleus ER
Plasma
Membrane
Mitochondria
Golgi

5. A variety of coat complexes participate in vesicle formation

6. 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.

7. 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

8. 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.

9. Vesicle Formation driven by coat assembly cargo is concentrated
SNAREs implicated
p24 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 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

11. 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

12. 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)

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 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)

16. 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

17. 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