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Mol Biol Cell. 2016 Aug 15;27(16):2590-7. doi: 10.1091/mbc.E16-04-0230. Epub 2016 Jul 6.

Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement.

Author information

1
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755-3844.
2
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755-3844 william.wickner@dartmouth.edu.

Abstract

In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion. 3) The Rab GTPase Ypt7 is essential in vivo but often dispensable in vitro. Here we report that changes in fatty acyl chain composition dramatically lower the density of SNAREs that are required for fusion. By providing more physiological lipids with a lower phase transition temperature, we achieved efficient fusion with SNARE concentrations as low as on the native organelle. Although fused proteoliposomes became unstable at elevated SNARE concentrations, releasing their content after fusion had occurred, reconstituted proteoliposomes with substantially reduced SNARE concentrations fused without concomitant lysis. The Rab GTPase Ypt7 is essential on both membranes for proteoliposome fusion to occur at these SNARE concentrations. Strikingly, it was only critical for Ypt7 to be GTP loaded on membranes bearing the R-SNARE Nyv1, whereas the bound nucleotide of Ypt7 was irrelevant on membranes bearing the Q-SNAREs Vam3 and Vti1.

PMID:
27385334
PMCID:
PMC4985260
DOI:
10.1091/mbc.E16-04-0230
[Indexed for MEDLINE]
Free PMC Article

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