et al., 1995) and in vacuole morphology (vam) screens for fragmented vacuole morphology (Wada et al., 1992), are also required for vacuole docking (Price et al., 2000). Docking triggers a brief release of calcium from the vacu- ole lumen (Peters and Mayer, 1998) which leads to vacu- ole association of a complex of calmodulin and protein phosphatase 1 (Peters et al., 1999), a dephosphorylation of a vacuolar target protein(s), release of the LMA1 cochap- erone (Xu et al., 1998), and membrane fusion. Despite this progress in defining the components that catalyze each stage of the reaction, the mechanism whereby the different stages are coupled is unknown. One of the most intriguing questions is how the priming of vac- uoles leads to their docking. Specifically, how does the disassembly of the cis-SNARE complex lead to the Ypt7p- dependent association of vacuoles, and what are the link- ing proteins? We now show that this function is fulfilled by a protein complex that contains Vam2/Vps41p and Vam6/ Vps39p, termed Vam2/6p, working as a vacuolar Rab/Ypt effector. Vam2p and Vam6p are localized to the vacuole as part of a large complex (Nakamura et al., 1997) that is required for the docking stage of homotypic vacuole fu- sion (Price et al., 2000). However, unlike other known Rab/Ypt effectors, Vam2/6p is initially bound to the cis- SNARE complex and is released from the SNAREs upon Sec18p-dependent priming. Vam2/6p can only then bind to Ypt7p to initiate docking, providing an important physi- cal and functional link between membrane priming and docking. Materials and Methods Biochemical Reagents Gdi1p was purified according to Haas et al. (1995). All inhibitors and anti- bodies were dissolved or dialyzed in PS buffer (20 mM Pipes-KOH, pH 6.8, 200 mM sorbitol) unless stated otherwise. Antibody Production and Purification Antibodies against the Vam2 and Vam6 proteins were prepared as de- scribed in Price et al. (2000). Assay for Release of Vam2/Vam6p Vacuoles from S. cerevisiae BJ3505 (wild-type or with hemagglutinin [HA]-tagged Vam6p) were incubated under standard fusion reaction con- ditions for 0–90 min. The reactions were then diluted fivefold with PS buffer and the vacuoles sedimented by centrifugation (14,000 g, 5 min, 48C). Proteins in the supernatant were precipitated by the addition of TCA to a final concentration of 15% (vol/vol), washed with ice-cold ace- tone, and analyzed by SDS-PAGE and immunoblotting. For some experiments, the chromosomal VAM6 gene was replaced with a HA epitope–tagged version of the VAM6 gene by recombination with a plasmid cassette containing the HA-tagged VAM6 gene. A 1.2-Kb Kpn1-EcoRI fragment of the VAM6 gene containing six copies of the HA epitope at the 59 end of the coding sequence (a gift from Dr. Yoh Wada, Osaka University, Osaka, Japan) was cloned into pRS306. This construct was then linearized at the MscI site in the VAM6 coding sequence and transformed into S. cerevisiae BJ3505. Transformants were analyzed for ex- pression of HA-Vam6p by SDS-PAGE followed by immunoblotting with the anti-HA mAb 12CA5 (Boehringer) and antiserum against Vam6 pro- tein. HA-tagged VAM6 rescues the fragmented vacuole phenotype of vam6D yeast (Nakamura et al., 1997), and fusion of HA-Vam6p vacuoles in vitro is indistinguishable from that of wild-type vacuoles (data not shown). Immunoprecipitations Anti-Vam2p, anti-Ypt7p, and anti-Vam3p were cross-linked to protein A–Sepharose CL-4B (Amersham Pharmacia Biotech) using dimethylpi- melimidate (Harlowe and Lane, 1988). Vacuoles (100 mg) were solubilized in 200 ml of solubilization buffer (20 mM Hepes-KOH, pH 7.4, 150 mM NaCl, 10% [wt/vol] glycerol, 1% [vol/vol] Triton X-100) with protease inhibitor cocktail (Haas, 1995) for 10 min on ice. Insoluble material was removed by centrifugation (14,000 g, 10 min, 48C). The supernatant was then incubated with protein A–Sepharose immobilized antibodies for 2 h at 48C in a final volume of 500 ml of solubilization buffer. The beads were washed twice with solubilization buffer and bound proteins were eluted with 0.1 M glycine, pH 2.5. The eluates were then neutralized by the addition of 0.1 M Tris-HCl, pH 9.0, and analyzed by SDS-PAGE and immunoblotting. Sucrose Velocity Gradients Vacuoles were sedimented (14,000 g, 10 min, 48C) and solubilized for 10 min on ice in PS solubilization buffer, which is PS buffer containing 1% (vol/vol) Triton X-100 and protease inhibitor cocktail (Haas, 1995). Insol- uble material was removed by sedimentation (14,000 g, 10 min, 48C). The vacuole extract was layered onto a 12-ml gradient of 10–40% (wt/vol) su- crose in PS buffer with 1% (vol/vol) Triton X-100 and centrifuged (38,000 rpm, 6 h, 48C; Beckman SW-41 rotor). Fractions were analyzed by SDS- PAGE followed by immunoblotting. BSA (4S), a2-macroglobulin (20S), and ferritin (65S) were centrifuged in a parallel gradient and analyzed by SDS-PAGE and staining with Coomassie brilliant blue. Results Association of Vam2/6p with SNAREs In accord with earlier studies (Nakamura et al., 1997), Vam2p and Vam6p are associated with each other as part of a large complex, as assayed in Triton X-100 extracts of vacuoles by coimmunoprecipitation (Fig. 1 A, lane 2) and cosedimentation at 65S in sucrose velocity gradients (Fig. 1 B). Gradient fractions were also assayed for the SNARE proteins Nyv1p and Vam3p. Surprisingly, although the cis- SNARE complex (Ungermann et al., 1999) was labile un- der these conditions, a small fraction of these SNAREs cosedimented with Vam2/6p (Fig. 1 B). This was con- firmed directly by observing a coimmunoprecipitation of Vam2p by anti-Vam3p (Fig. 2 A, lane 1), but not by con- trol IgG (Fig. 2 A, lane 4). This association was seen with other members of the SNARE complex, as antibody to the v-SNAREs Vti1p or Ykt6p immunoprecipitate Vam2p (Fig. 2 B, lanes 2 and 3) at least as efficiently as does anti- body to Vam3p (Fig. 2 B, lane 1). Vacuole cis-SNARE complexes are dissociated by the priming activity of Sec18p (Ungermann et al., 1999). We find that Vam2/6p is released from its association with the cis-SNARE complex during priming. Coimmunoprecipita- tion of Vam2p with Vam3p is diminished 77% upon incu- bation at 278C with ATP (Fig. 2 A, lane 2), and this release is completely blocked by antibody to the priming catalyst Sec18p (Fig. 2 A, lane 3) but not by ligands that block steps other than priming (data not shown). Not only do SNAREs cosediment (Fig. 1 B) and coimmunoprecipitate (Fig. 2, A and B) with Vam2/6p, but disruption of the cis- SNARE complex through priming the vacuoles (Fig. 3) causes a shift of the 65S Vam2/6p to a slower sedimenting species of 38S. As Vam2/6p is a peripheral membrane complex (Nakamura et al., 1997), this loss of its association with integral membrane SNAREs, as well as its involve- ment in the docking reaction (Price et al., 2000), raises the question of the identity of its subsequent physical and functional associations on the vacuole membrane. The Journal of Cell Biology, Volume 148, 2000 1232