Vacuole membrane fusion

Abstract

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. Δvph1 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.