Yeast Vacuole Fusion Assay

1
Yeast Vacuole Fusion Assay
Haas et al. (1994) J. Cell Biol. 126, 87-97
2
In Vitro Reconstitution of Yeast Vacuole Fusion
Haas et al. (1994) J. Cell Biol. 126, 87-97
3
Released Sec17p (aSNAP) Is No Longer Needed for
Fusion
Start Fusion Reaction
Conclusion of this and similar assays Action of
a-SNAP and NSF precedes Fusion !
Mayer et al. (1996) Cell 85, pp 83-94
4
Are SNAREs Sufficient for Fusion?
5
(No Transcript)
6
Regulated Secretion at the Neuronal Synapse
7
Purification of Neuronal SNAREs from E. coli
Weber et al. (1998) Cell 92, pp759-772
8
Reconstitution of SNAREs into Lipid-Vesicles
9
SNARE-Containing Vesicles After Density Gradient
Weber et al. (1998) Cell 92, pp759-772
10
Fluorescence Resonance Energy Transfer (FRET)
11
(No Transcript)
12
FRET Efficiency
R0 Förster Radius (E50) R Distance between
Donor and Acceptor Fluorophore
13
(No Transcript)
14
Fusion of v-SNARE and t-SNARE Vesicles
Weber et al. (1998) Cell 92, pp759-772
15
Conversion to Rounds of Fusion of v-SNARE vesicles
16
SNARE Mediated Fusion Is Specific
17
(No Transcript)
18
Comparison of Structures of Viral and Cellular
Fusion Proteins
19
Dissociation of SNAREs after Fusion
20
Proposed Life-Cycle of SNAREs
Weber et al. (2000) J. Cell Biol. 149, pp1063-1072
21
Are SNAREs Major Determinants of Vesicle
Targeting ?
Cons
Not Enough SNAREs for Every Transport
Step Non-Cognate SNARE-Complexes Can Form In
Vitro Non-Cognate SNARE-Complexes Are Very
Stable
22
(No Transcript)
23
(No Transcript)
24
Intracellular Localization of Rab-Proteins
Zerial and McBride (2001) Nat. Rev. Mol. Cell
Bio. 2, pp 107-119
25
Possible Role for Rab Proteins
(GAP)