Mitochondria Plasma Membrane Nucleus Lysosome ER Golgi.

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