Figure 3. Vacuole priming dissociates the Vam2/6p complex. BJ3505 wild-type vacuoles (300 mg) were incubated at 278C for 10 min with or without Mg21/ATP in standard fusion reactions. Vacuoles were then sedimented, solubilized in PS buffer contain- ing 1% Triton X-100, and analyzed on sucrose velocity gradients as in Fig. 1. with Ypt7p in the cell, we examined the localization of Vam6p in a ypt7D strain. Purified vacuoles that lack Ypt7p had very little Vam6p (Fig. 5, lane 4) in comparison with wild-type vacuoles (Fig. 5, lane 3), whereas the amount of the vacuole marker Pho8p is unaffected by the ypt7D mu- tation. The amount of Vam6p is unchanged in whole cell lysates of ypt7D cells in comparison with wild-type (Fig. 5, lanes 1 and 2). Therefore, the ypt7D mutation affects the vacuolar localization of Vam6p but not its expression, in- dicating a functional relationship between these compo- nents. In contrast, the amount of Ypt7p on the vacuoles is unaffected by the vam2D and vam6D mutations (see Fig. 2 of Price et al., 2000, this issue), indicating that Ypt7p has a primary role in vacuolar localization and that Vam2p and Vam6p are dependent on Ypt7p. In accord with Ypt7p being required for the vacuolar lo- calization of Vam6p in vivo, extraction of Ypt7p or inhibi- tion of its function in vitro releases Vam2 and Vam6 pro- teins from the vacuole membrane. When vacuoles are incubated under standard fusion conditions, ,1% of Vam2 or Vam6 protein is released into the reaction super- natant after sedimentation (Fig. 6 A, lane 2; supernatant of a standard fusion reaction). Vam2/6p release is strikingly enhanced by the addition of yeast Gdi1p (Sec19p), an in- hibitor of Rab GTPases that binds and extracts the GDP- bound form of these proteins from membranes (Novick and Zerial, 1997). As seen in Fig. 6 A, lane 4, the 22% of Vam2p and Vam6p that was released from the vacuoles upon Ypt7p release by Gdi1p is recovered in the reaction supernatant. The integral membrane protein Pho8p is not found in any of the reaction supernatants, indicating that the appearance of Vam2p and Vam6p in the supernatant is not due to fragmentation of the vacuoles. The Vam6p that is extracted by Gdi1p is still coimmunoprecipitated by anti-Vam2p (Fig. 1 A, lane 4), although the extracted com- plex sediments more slowly than Vam2/6p in association Figure 4. Association of Vam2/6p with Ypt7p. A. Vam6p is asso- ciated with Ypt7p on isolated vacuoles. BJ3505 HA-Vam6p vacu- oles were solubilized in buffer containing 1% Triton X-100 as described in Materials and Methods, and proteins were immuno- precipitated from the detergent extracts with preimmune serum or antiserum to Ypt7p. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with anti-HA mAb 12CA5 and anti-Ypt7p. (B) The 38S Vam2/6p complex binds to GST–Ypt7p: GTPgS. BJ3505 vacuoles (650 mg) were sedimented (13,000 rpm, 10 min, 48C; Microfuge, then incubated in a 1003 scale fusion re- action (Haas, 1995) with salt adjustment buffer for 10 min at 278C. Primed vacuoles were then sedimented as above and resus- pended twice in 1 ml PS buffer followed by sedimentation. Primed vacuoles, as well as a separate aliquot of unprimed vacuoles (650 mg), which had also been sedimented and resuspended, were solu- bilized in 650 ml PS solubilization buffer and fractionated on su- crose gradients as described in Materials and Methods. Vam2/6p peak fractions from unprimed (65S) and primed (38S) vacuoles (see Fig. 3) were brought to final concentration of 150 mM NaCl, 10% glycerol, and 13 protease inhibitor cocktail (Haas, 1995), and incubated for 2 h at 48C in the presence of 12 mg of glu- tathione–Sepharose-bound GST–Ypt7p or GST–Ypt1p. (Fusion protein preparation: The fusion proteins were preincubated in PS solubilization buffer containing 2 M NaCl, 10% glycerol, 1 mM DTT, 20 mM EDTA, and 5 mM GDP [PS elution buffer] for 20 min at room temperature with 40 ml (packed volume) glu- tathione–Sepharose 4B beads [Amersham Pharmacia Biotech]. Beads were sedimented [1 min, 13,000 rpm, 48C] and resuspended four times as above in 1 ml of PS solubilization buffer containing 150 mM NaCl and 10% glycerol, then incubated in 25 ml of 1.23 PS buffer containing 36 mM TrisCl, pH 8.0, 180 mM KCl, 0.6 mM MnCl2, 6 mM MgCl2, 0.13 protease inhibitor cocktail, 1 mM DTT, and either 4 mM GTPgS, 4 mM GDP, or no nucleotide [2] for 30 min at 278C). After the incubation of peak fractions and fu- sion protein beads, beads were collected and suspended twice in 1 ml PS solubilization buffer. Bound proteins were eluted with 500 ml PS elution buffer for 15 min at room temperature and sepa- rated from the beads by sedimentation as above. The elutate was TCA-precipitated, the precipitate suspended in 1 ml 80% acetone and collected by sedimentation, and analyzed by SDS-PAGE and immunoblotting with antisera to Vam6p and Vam3p. with the cis-SNAREs (data not shown). Thus, Vam2p and Vam6p remain together throughout priming and extrac- tion by loss of their Ypt7p anchor. We also observed that some Vam2p is covalently modified upon release and mi- The Journal of Cell Biology, Volume 148, 2000 1234