Figure 5. Vam6p is lost from the vacuole in ypt7D cells. BJ3505 wild-type and ypt7D cultures (1 l) were grown to mid-log phase. Portions (50 ml) of each were centrifuged and the cells were re- suspended in buffer containing 2% (vol/vol) Triton X-100, 1% (wt/vol) SDS, 0.1 M NaCl, 1 mM EDTA, 1 mM PMSF, and 10 mM TrisCl, pH 8.0. Cells were broken by vortexing in the presence of acid-washed glass beads. Glass beads and cell debris were re- moved by centrifugation (48C, 1 min, 14,000 g). The remainder of the culture was used for vacuole purification (see Materials and Methods). Lysed cell supernatant from equivalent amounts of cells or equivalent amounts of vacuolar protein (15 mg) were ana- lyzed by SDS-PAGE and immunoblotting with antisera against Vam6p and Pho8p. grates at a slightly lower mobility, though the nature of this modification and its significance have not yet been de- termined. The removal of Ypt7p from the vacuole by Gdi1p is dose-dependent and there is a corresponding re- lease of Vam6p (data not shown). However, measurement of release as a function of reaction time shows that Ypt7p is removed very rapidly from the vacuoles by Gdi1p, whereas the loss of Vam6p occurs gradually (Fig. 6 B). Ypt7p is specifically required for the retention of Vam6p on the vacuole, since addition of inhibitory anti-Ypt7p IgG (Haas et al., 1995) causes 27% release of Vam6p (Fig. 6 A, lane 6), and is thus as effective as Gdi1p in releasing Vam6p, whereas preimmune IgG has no effect (Fig. 6 A, lane 8). However, unlike Gdi1p, anti-Ypt7p does not re- move Ypt7p from the vacuole membrane (Fig. 6 A, lanes 5 and 6). Therefore, it is the loss of full Ypt7p function and not its removal from the vacuole membrane that causes re- lease of the Vam2 and Vam6 proteins. Although ATP is not required for the extraction of Ypt7p by Gdi1p, ATP is required for the release of Vam2p and Vam6p by either Gdi1p or anti-Ypt7 (Fig. 6 A, lanes 2–6). To determine whether ATP is needed at the same time as Gdi1p to promote Vam2/6p release, a two-stage reaction was performed. Vacuoles were first incubated with Gdi1p to remove Ypt7p, then reisolated to remove the Gdi1p. The vacuoles were then incubated with or with- out ATP and sedimented to allow assay of released Vam6p (Fig. 6 C). ATP alone in the second incubation is sufficient to extract Vam6p after Gdi1p treatment in the first incubation (Fig. 6 C, lane 4). However, whereas com- plete release of Ypt7p is achieved in the absence of ATP in the first 10-min incubation (Fig. 6 C, lane 3), little Vam6p is removed in this short incubation even when both Gdi1p and ATP are present (Fig. 6 C, lane 5). There- fore, the ATP requirement for the release of Vam6p can- not be simply due to effects it might have on Ypt7p, and may not simply reflect a need for priming, which is usually complete by 10 min (Mayer et al., 1996; Price et al., accom- Figure 6. Release of Vam2/6p from the vacuole. (A) Vam2/6p release. BJ3505 HA-Vam6p vacuoles (see Materials and Meth- ods) were incubated under standard fusion conditions with or without ATP at 278C. Reactions were supplemented with control buffer, Gdi1p (64 mg/ml), IgG against Ypt7p, or preimmune IgG. After 90 min, the reactions were diluted fivefold with PS buffer and the vacuoles sedimented (5 min, 14,000 g, 48C). The superna- tants were then TCA-precipitated and analyzed by SDS-PAGE and immunoblotting with anti-HA mAb 12CA5 or antisera against Vam2p, Ypt7p, and Pho8p. Lanes 5–8 of the Vam2p panel were omitted because the immunoblot of Vam2p is ob- scured by the rabbit IgG added to the reactions. (B) Vam6p is re- leased from the vacuole after the release of Ypt7p. BJ3505 vacu- oles were incubated under standard fusion conditions with ATP at 278C in the presence of Gdi1p (64 mg/ml). At the indicated times, reactions were placed on ice. After sedimenting as above, supernatants were analyzed by SDS-PAGE and immunoblotting with antibodies to Vam6p and Ypt7p. (C) The ATP requirement for Vam6p release is unrelated to the release of Ypt7p. BJ3505 vacuoles were incubated at 278C for 10 min with ATP and/or Gdi1p (64 mg/ml) as indicated. The reactions were then diluted 10-fold with PS buffer and the vacuoles reisolated to remove ATP and Gdi1p. Vacuoles were then resuspended in reaction buffer with or without ATP as indicated and incubated at 278C for an additional 80 min. Reactions were then analyzed for re- lease of Vam6p and Ypt7p as above. panying manuscript). The role of ATP in Gdi1p-mediated release of Vam2/6p remains to be determined. Sec18-primed Vacuoles must Dock to Retain the Vam2/6 Complex Since Ypt7p supports vacuole docking (Mayer and Wick- Price et al. Yeast Vacuole Fusion 1235