Vacuole membrane fusion: V(0) functions after trans-SNARE pairing and is coupled to the Ca(2+)-releasing channel

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 V(0) 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 V(0) sectors in vacuole fusion. V(0) functions in fusion and proton translocation could be experimentally separated via the differential effects of mutations and inhibitory antibodies. Inactivation of the V(0) 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 Ca(2+), but they did not fuse. The Ca(2+)-releasing channel appears to be tightly coupled to V(0) because inactivation of Vph1p by antibodies blocked Ca(2+) release. Vph1 deletion on only one fusion partner sufficed to severely reduce fusion activity. The functional requirement for Vph1p correlates to V(0) transcomplex formation in that both occur after docking and Ca(2+) release. These observations establish V(0) as a crucial factor in vacuole fusion acting downstream of trans-SNARE pairing.