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PMID: 11118206 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Sequential action of two GTPases to promote vacuole docking and fusion.

The EMBO journal ·Vol. 19 ·No. 24 ·2000-12-15 ·Pages 6713-20

Eitzen G, Will E, Gallwitz D, Haas A, Wickner W

Abstract

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), GTPgammaS 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 Ca(2+)-triggered cascade that leads to fusion. However, in the absence of Ypt7p, fusion is still sensitive to GTPgammaS 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.

MeSH Terms
Calcium/physiology Calcium Signaling Fungal Proteins/metabolism Genotype Guanine Nucleotide Dissociation Inhibitors/metabolism Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology Guanosine Diphosphate/metabolism Guanosine Triphosphate/metabolism Guanylyl Imidodiphosphate/pharmacology Kinetics Membrane Fusion/drug effects,physiology Protein Binding Saccharomyces cerevisiae/genetics,physiology,ultrastructure Saccharomyces cerevisiae Proteins Vacuoles/drug effects,physiology,ultrastructure rab GTP-Binding Proteins/metabolism
Chemicals
Fungal Proteins GDP dissociation inhibitor 1 Guanine Nucleotide Dissociation Inhibitors Saccharomyces cerevisiae Proteins Guanosine Diphosphate Guanylyl Imidodiphosphate Guanosine 5'-O-(3-Thiotriphosphate) Guanosine Triphosphate YPT7 protein, S cerevisiae rab GTP-Binding Proteins Calcium
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Eitzen G
Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3844, USA.
Will E
Gallwitz D
Haas A
Wickner W
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1093/emboj/19.24.6713
Published
2000-12-15
Pages
6713-20
Language
English
Region
England
NLM ID
8208664
PMCID
PMC305897
Subset
IM
External Links
PubMed source DOI: 10.1093/emboj/19.24.6713 PMC Full Text View journal details
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