Abstract: Yeast vacuole fusion requires SNAREs, the Rab GTPase Ypt7p, vacuolar lipids, Sec17p and Sec18p, and the HOPS complex. HOPS is a multisubunit protein with direct affinities for SNAREs, vacuolar lipids and the GTP-bound form of Ypt7p; each of these affinities contributes to HOPS association with the organelle. Using all-purified components, we have reconstituted fusion, but the Rab Ypt7p was not required. We now report that phosphorylation of HOPS by the vacuolar kinase Yck3p blocks HOPS binding to vacuolar lipids, making HOPS membrane association and the ensuing fusion depend on the presence of Ypt7p. In accord with this finding in the reconstituted fusion reaction, the inactivation of Ypt7p by the GTPase-activating protein Gyp1-46p only blocks the fusion of purified vacuoles when Yck3p is present and active. Thus, while Ypt7p may contribute to other fusion functions, its central role is to bind HOPS to the membrane.