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© The Rockefeller University Press, 0021-9525/2003/7/211 $5.00
The Journal of Cell Biology, Volume 162, Number 2, 211-222

Vacuole membrane fusion

Martin J. Bayer, Christoph Reese, Susanne Bühler, Christopher Peters and Andreas Mayer

Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, 72076 Tübingen, Germany

Address correspondence to Andreas Mayer, Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstr. 37-39, 72076 Tübingen, Germany. Tel.: 49-7071-601850. Fax: 49-7071-601455. E-mail: Andreas.Mayer{at}Tuebingen.mpg.de

Pore models of membrane fusion postulate that cylinders of integral membrane proteins can initiate a fusion pore after conformational rearrangement of pore subunits. In the fusion of yeast vacuoles, V-ATPase V₀ sectors, which contain a central cylinder of membrane integral proteolipid subunits, associate to form a transcomplex that might resemble an intermediate postulated in some pore models. We tested the role of V₀ sectors in vacuole fusion. V₀ functions in fusion and proton translocation could be experimentally separated via the differential effects of mutations and inhibitory antibodies. Inactivation of the V₀ subunit Vph1p blocked fusion in the terminal reaction stage that is independent of a proton gradient. vph1 mutants were capable of docking and trans-SNARE pairing and of subsequent release of lumenal Ca²⁺, but they did not fuse. The Ca²⁺-releasing channel appears to be tightly coupled to V₀ because inactivation of Vph1p by antibodies blocked Ca²⁺ release. Vph1 deletion on only one fusion partner sufficed to severely reduce fusion activity. The functional requirement for Vph1p correlates to V₀ transcomplex formation in that both occur after docking and Ca²⁺ release. These observations establish V₀ as a crucial factor in vacuole fusion acting downstream of trans-SNARE pairing.

Key Words: membrane fusion; vacuoles; SNAREs; V-ATPase; calcium

^* Abbreviations used in this paper: BCECF, 2'7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein; FCCP, carbonylcyanide-4-trifluormethoxyphenylhydrazon; pmf, proton motive force.

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