G-protein ligands inhibit in vitro reactions of vacuole inheritance.
During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures. One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole. This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. J. Cell Biol. 119:1469-1479). We now report a simple and reliable assay to quantify vacuole-to-vacuole fusion in vitro. This assay is based on the maturation and activation of vacuole membrane-bound pro-alkaline phosphatase by vacuolar proteinase A after vacuole-to-vacuole fusion. In vitro fusion allowed maturation of 30 to 60% of pro-alkaline phosphatase. Vacuoles prepared from a mutant defective in vacuole inheritance in vivo (vac2-1) were inactive in this assay. Vacuole fusion in vitro required a vacuole membrane potential. Inhibition by nonhydrolyzable guanosine derivatives, mastoparans, and benzalkonium chloride suggest that GTP-hydrolyzing G proteins may play a key role in the in vitro fusion events.
Molecular Biology Institute, University of California, Los Angeles 90024-1570.
A
A
Haas
B
B
Conradt
W
W
Wickner
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D001548:Benzalkonium Compounds / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006151:Guanosine / Q000031:analogs & derivatives; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D018942:Macrolides; D008561:Membrane Fusion / Q000187:drug effects; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D010455:Peptides; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D014617:Vacuoles / Q000187:drug effects / Q000502:physiology*; D014862:Wasp Venoms / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D001548:Benzalkonium Compounds; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D018942:Macrolides; D010455:Peptides; D014862:Wasp Venoms; D006151:Guanosine; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C037500:mastoparan; C040929:bafilomycin A1; D019204:GTP-Binding Proteins; D009355:Neomycin