Sequential action of two GTPases to promote vacuole docking and fusion

Homotypic vacuole fusion occurs by sequential priming, docking and fusion r eactions. Priming frees the HOPS complex (Vps 11, 16, 18, 33, 39 and 41) to activate Ypt7p for docking. Here we explore the roles of the GDP and GTP s tates of Ypt7p using Gdi1p (which extracts Ypt7:GDP), Gyp7p (a GTPase-activ ating protein for Ypt7p:GTP), GTP gammaS or GppNHp (non-hydrolyzable nucleo tides), and mutant forms of Ypt7p that favor either GTP or GDP states. GDP- bound Ypt7p on isolated vacuoles can be extracted by Gdi1p, although only t he GTP-bound state allows docking. Ypt7p is converted to the GTP-bound stat e after priming and stably associates with HOPS. Gyp7p can cause Ypt7p to h ydrolyze bound GTP to GDP, driving HOPS release and accelerating Gdi1p-medi ated release of Ypt7p. Ypt7p extraction does not inhibit the Ca2+-triggered cascade that leads to fusion. However, in the absence of Ypt7p, fusion is still sensitive to GTP gammaS and GppNHp, indicating that there is a second specific GTPase that regulates the calcium flux and hence fusion. Thus, tw o GTPases sequentially govern vacuole docking and fusion.