Excess vacuolar SNAREs drive lysis and Rab bypass fusion
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Edited by Thomas C. Sudhof, University of Texas Southwestern Medical Center, Dallas, TX, and approved June 28, 2007 (received for review May 23, 2007)
Abstract
Although concentrated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) drive liposome fusion and lysis, the fusion of intracellular membranes also requires Rab GTPases, Rab effectors, SM proteins, and specific regulatory lipids and is accompanied by little or no lysis. To rationalize these findings, we generated yeast strains that overexpress all four vacuolar SNAREs (4SNARE ++). Although vacuoles with physiological levels of Rab, Rab effector/SM complex, and SNAREs support rapid fusion without Rab- and SNARE-dependent lysis, vacuoles from 4SNARE ++ strains show extensive lysis and a reduced need for the Rab Ypt7p or regulatory lipids for fusion. SNARE overexpression and the addition of pure homotypic fusion and vacuole protein sorting complex (HOPS), which bears the vacuolar SM protein, enables ypt7Δ vacuoles to fuse, allowing direct comparison of Rab-dependent and Rab-independent fusion. Because 3- to 40-fold more of each of the five components that form the SNARE/HOPS fusion complex are required for vacuoles from ypt7Δ strains to fuse at the same rate as vacuoles from wild-type strains, the apparent forward rate constant of 4SNARE/HOPS complex assembly is enhanced many thousand-fold by Ypt7p. Rabs function in normal membrane fusion by concentrating SNAREs, other proteins (e.g., SM), and key lipids at a fusion site and activating them for fusion without lysis.
Footnotes
- †To whom correspondence should be addressed. E-mail: bill.wickner{at}dartmouth.edu
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↵Author contributions: *V.J.S. and Y.J. are co-first authors; V.J.S., Y.J., and W.W. designed research; V.J.S. and Y.J. performed research; and V.J.S., Y.J., and W.W. wrote the paper.
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This Feature Article is part of a series identified by the Editorial Board as reporting findings of exceptional significance.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission.
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See Commentary on page 13541.
- Abbreviations:
- SNARE,
- soluble N-ethylmaleimide-sensitive factor attachment protein receptor;
- HOPS,
- homotypic fusion and vacuole protein sorting complex;
- sGFP,
- Superglow GFP.
- © 2007 by The National Academy of Sciences of the USA
