THE GTPASE YPT7P OF SACCHAROMYCES-CEREVISIAE IS REQUIRED ON BOTH PARTNER VACUOLES FOR THE HOMOTYPIC FUSION STEP OF VACUOLE INHERITANCE

In the budding yeast Saccharomyces cerevisiae, vacuoles are inherited by the projection of vesicles and tubules from the mother-cell vacuole into the growing daughter cell during the S phase. These vesicles the n fuse and form the daughter-cell organelle. We have described previou sly in vitro reactions of the formation of vacuole-derived segregation structures and of vacuole-vacuole fusion. Homotypic vacuole fusion re quires cytosol, ATP and a physiological temperature, and is sensitive to GTPase inhibitors. These reactions are divisible into early stages which require ATP and cytosol, and late stages which require neither. Here, we report that Ypt7p, a ras-like GTPase implicated previously in endocytosis in yeast, is largely localized to the vacuole and is requ ired on both partners during the in vitro vacuole fusion reaction, The in vitro fusion reaction is inhibited either by Gdi1p, which extracts the GDP-bound form of ras-like GTPases from membranes, or by antibodi es specific for Ypt7p. The presence of anti-Ypt7p during the early sta ges of the reaction inhibits the development of cytosol- and ATP-indep endent intermediates, Although cytosol and ATP are no longer needed fo r the late stage of vacuole inheritance in vitro, the inhibition of th is late stage by anti-Ypt7p or Gdi1p requires the continued presence o f ATP and cytosol, Ypt7p is the first GTPase for which a direct role i n organelle inheritance has been established.