Figure 7. Sec18-primed vacuoles must dock to retain Vam6p. BJ3505 HA-Vam6p vacuoles were incubated with or without ATP with control buffer, antibody against Sec18p, or preimmune IgG on ice for 10 min. Lane 6 was supplemented with Sec18 pro- tein (100 mg/ml). Lane 2 received Gdi1p (64 mg/ml). Lanes 3–7 were then diluted 20-fold with reaction buffer with ATP where indicated. All reactions were then incubated at 278C for 90 min and analyzed for HA-Vam6p release. ner, 1997; Ungermann et al., 1998b), we asked whether inhibition of docking itself would influence Vam2/6p localization. 20-fold dilution of the vacuoles, a non- pharmacological treatment that prevents vacuole docking (Mayer and Wickner, 1997), promotes Vam6p release from the vacuoles (Fig. 7, lane 4). Like the Vam6p release promoted by Ypt7p inhibitors, ATP is required for Vam6p release upon dilution (Fig. 7, lane 3 vs. lane 4). Dilution does not cause release of Ypt7p (data not shown). This Vam6p release requires the normal priming action of Sec18p, as addition of anti-Sec18p blocks Vam6p release upon dilution of the fusion reaction (Fig. 7, lane 5), whereas preimmune IgG had no effect (Fig. 7, lane 7). The anti-Sec18p blockage of this reaction was relieved by the addition of excess Sec18 protein (Fig. 7, lane 6), demon- strating that the effect of the antibody and the promotion of Vam6p release is specifically due to the activity of Sec18p. Therefore, after priming releases Vam2/6p from its association with the cis-SNARE complex, vacuole docking must occur to avoid Vam2/6p release. Discussion Our current studies suggest a working model (Fig. 8) of how Vam2/6p links priming and docking. In this model, Sec18p-dependent priming releases Vam2/6p from its as- sociation with the cis-SNARE complex (Fig. 8 A), freeing it to interact with Ypt7p (Fig. 8 B) and other docking com- ponents. After priming, Vam2/6p is needed for docking (Fig. 8 C) and docking is needed for the continued vacuole association of Vam2/6p (Figs. 5–7), suggesting that Vam2/6p is at the docking site (Fig. 8 C). Since Vam2/6p is required on both partner vacuoles (Price et al., 2000), Vam2/6p complexes from apposing vacuoles may contact one an- other during docking, although their function may also re- quire other associated proteins. Vam2/6p may promote trans-SNARE pairing (Fig. 8 D) largely through working together with Ypt7p to mediate tethering. In this model, Vam2/6p regulates docking through its cycle of associa- tions. It remains on the vacuole in vivo and during stan- dard fusion reactions, yet after priming, even dilution is sufficient to cause its release. The inability of Ypt7p, Figure 8. Working model for the coupling of vacuolar priming, docking, and trans-SNARE pairing. v, t, and s symbolize the v-SNAREs (Nyv1p, Vti1p, and Ykt6p), the t-SNARE Vam3p, and the SNAP-25 homologue Vam7p, respectively. Y7, Ypt7p; 2/6, Vam2/6 complex. See text for details. which is not released upon dilution, to anchor Vam2/6p to diluted primed vacuoles suggests that Vam2/6p is directly involved in trans interactions. Each aspect of this model (Fig. 8) is supported by our current studies. Vam2/6p is initially in a complex with SNAREs, as judged by cosedimentation (Fig. 1 B) and coimmunoprecipitation (Fig. 2). We find that priming, as it frees Vam2/6p from its association with SNAREs (Fig. 2 A), releases a 38S subcomplex (Fig. 3), which has gained an ability to associate with Ypt7p that was not seen before priming (Fig. 4 B). Functional in vitro studies are in com- plete accord with the transfer of Vam2/6p from association with SNAREs to Ypt7p, as Vam2/6p does not complete its function until docking (Price et al., 2000) and extraction of Ypt7p by Gdi1p, or its inhibition by anti-Ypt7p disrupts the vacuole association of Vam2/6p (Fig. 6). Finally, the The Journal of Cell Biology, Volume 148, 2000 1236