Sequential action of two GTPases to promote vacuole docking and fusion

• Published in: The EMBO journal Vol. 19; no. 24; pp. 6713 - 6720
• Main Authors: Eitzen, Gary, Will, Elke, Gallwitz, Dieter, Haas, Albert, Wickner, William
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 15.12.2000; Blackwell Publishing Ltd; Oxford University Press
• Subjects: Calcium - physiology; Calcium Signaling; Fungal Proteins - metabolism; Genotype; GTPase; Guanine Nucleotide Dissociation Inhibitors - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Guanosine Diphosphate - metabolism; Guanosine Triphosphate - metabolism; Guanylyl Imidodiphosphate - pharmacology; HOPS; Kinetics; Membrane Fusion - drug effects; Membrane Fusion - physiology; Protein Binding; rab GTP-Binding Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - physiology; Saccharomyces cerevisiae - ultrastructure; Saccharomyces cerevisiae Proteins; vacuole docking; vacuole fusion; Vacuoles - drug effects; Vacuoles - physiology; Vacuoles - ultrastructure; Ypt7p
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.1093/emboj/19.24.6713

Homotypic vacuole fusion occurs by sequential priming, docking and fusion reactions. 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 states of Ypt7p using Gdi1p (which extracts Ypt7:GDP), Gyp7p (a GTPase‐activating protein for Ypt7p:GTP), GTPγS or GppNHp (non‐hydrolyzable nucleotides), 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 the GTP‐bound state allows docking. Ypt7p is converted to the GTP‐bound state after priming and stably associates with HOPS. Gyp7p can cause Ypt7p to hydrolyze bound GTP to GDP, driving HOPS release and accelerating Gdi1p‐mediated 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 GTPγS and GppNHp, indicating that there is a second specific GTPase that regulates the calcium flux and hence fusion. Thus, two GTPases sequentially govern vacuole docking and fusion.