Figure 1. Physical associations of Vam2p and Vam6p. (A) Vam2p and Vam6p form a stable complex. BJ3505 HA-Vam6p vacuoles were solubilized with buffer containing 1% Triton X-100 or incubated at 278C in a complete fusion reaction contain- ing Gdi1p (64 mg/ml) for 90 min. Detergent extracts and release reaction supernatants were immunoprecipitated with antibody against Vam2p or preimmune antibody as described in Materials and Methods. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with anti-HA mAb 12CA5. (B) Sucrose velocity gradients. BJ3505 vacuoles (500 mg) were solubilized in 400 ml of PS solubilization buffer and analyzed on 10–40% su- crose gradients as described in Materials and Methods. Gradient fractions were analyzed by SDS-PAGE and immunoblotting with antisera against Vam2p, Vam6p, Nyv1p, and Vam3p. Immuno- blots were then quantified by scanning densitometry and plotted as a percent of the maximum signal in the peak fraction. Vam2/6p Physically Associates with Ypt7p after Priming Since both Vam2/6p and Ypt7p are essential for docking (Mayer and Wickner, 1997; Price et al., 2000), we asked whether Vam2/6p and Ypt7p are directly associated. We find that antibody to Ypt7p will cross-immunoprecipitate z13% of the Vam6p from vacuole detergent extracts (Fig. 4 A). Moreover, the GTP form of purified recombinant glutathione S-transferase (GST)–Ypt7p will bind the com- plex containing Vam6p (Fig. 4 B, lane 3) and Vam2p (data not shown) from the 38S gradient-purified fractions (Fig. 3) of Vam2/6p complex from primed vacuoles. This associ- Figure 2. Vam2/6p is associated with the cis-SNARE complex. (A) Association of Vam2p with Vam3p. BJ3505 vacuoles were incubated in 103 scale fusion reactions at 278C for 10 min with or without ATP. Lane 3 was supplemented with antibody to Sec18p. Vacuoles were solubilized in buffer containing 1% Triton X-100 as described in Materials and Methods, and proteins immunopre- cipitated from the detergent extracts with antibody to Vam3p. Immunoprecipitates were analyzed by SDS-PAGE and immuno- blotting with antisera to the indicated vacuolar proteins. (B) Vam2p is associated with Vti1p and Ytk6p. An immunoblot of vacuole protein immunoprecipitates from Ungermann et al. (1999) was probed with antibody to Vam2p. ation is specific for the nucleotide (Fig. 4 B, lanes 2–4) and is not seen with the GTP form of the fusion protein of GST with Ypt1p (Fig. 4 B, lane 5), a closely related Rap/ Ypt protein required for ER to Golgi traffic. Without priming, the 65S Vam2/6p complex (Fig. 4 B, lane 6) does not associate with GST–Ypt7p:GTPgS (Fig. 4 B, lane 8) above background levels (Fig. 4 B, lanes 7–10). These re- sults indicate that the Vam2/6p complex acquires a direct affinity for Ypt7p after priming and establish that Vam2/ 6p is an important Ypt7p effector. Ypt7p Regulates the Localization of Vam2/6p to the Vacuole To investigate whether Vam2p and Vam6p also interact Price et al. Yeast Vacuole Fusion 1233