absence of Ypt7p due to the deletion of its gene leads to a loss of Vam2/6p from the vacuole fraction (Fig. 5). The 38S Vam2/6p complex from primed vacuoles will bind to GST–Ypt7p:GTPgS, as judged by recovery of Vam2p and Vam6p from the bound fraction (Fig. 4). Though little of the endogenous Ypt7p cosediments with Vam2/6p at 38S (data not shown), this may reflect a lim- ited binding affinity and high dilution during purification that is overcome in experiments that employ glutathione– Sepharose beads with high levels of bound recombinant GST–Ypt7p. Recent studies (Seals, D., G. Eitzen, W. Wickner, and A. Price, manuscript in preparation) have shown that this bound complex, and the 65S and 38S com- plexes from which it derives, also include the four class C VPS proteins, Vps11p, Vps16p, Vps18p, and Vps33p. However, we cannot infer from these data which subunit or subunits of the 38S complex directly interact with Ypt7p, and which are only associated because they are part of the complex but perform Rab/Ypt effector func- tions. Further studies will be required to determine these functional distinctions. Our current findings and the model that they suggest have several novel aspects. The cis-SNARE complex is seen to not just be a left-over from trans-SNARE com- plexes after membrane fusion is completed, but rather is embedded in a 65S complex, which has a vital signaling role, mediated by Vam2/6p, and triggered by the action of Sec18p and Sec17p. It will certainly be important to study the pathway and regulation of 65S complex assembly. Vam2/6p fulfills the definition of a Rab effector, yet it as- sociates with a cis-SNARE complex upstream of its trans- fer to Ypt7p. The action of Sec18p and Sec17p is required for this transfer, providing the first functional evidence linking the priming factors with Rab/Ypt GTPases and their effectors. It will be important to determine whether the association of Rab effectors with their cognate SNAREs in other systems is with cis- or trans-SNARE complexes and whether it occurs before or after Rab asso- ciation. Vam2/6p functions on the vacuole with the Rab GTPase Ypt7p as a site-specific element for docking. It is, in this regard, functionally analogous to the plasma membrane– localized exocyst complex in yeast and mammals (Bowser et al., 1992; TerBush et al., 1996; Kee et al., 1997; Finger et al., 1998; Grindstaff et al., 1998), the cis-Golgi–localized transport protein particle (TRAPP) complex in yeast (Sacher et al., 1998), and early endosomal autoantigen 1 (EEA1) proteins in homotypic endosome–endosome fu- sion (Stenmark et al., 1995; Christoforidis et al., 1999; McBride et al., 1999). In addition, we propose that it func- tions as a link between priming and docking and suggest that other Rab/Ypt effectors may also fulfill these func- tions. Indeed, these complexes share a number of features. Like the TRAPP and exocyst complexes purified from cy- tosol (Bowser and Novick, 1988; Sacher et al., 1998), the soluble Vam2/6p complex released by Gdi1p migrates at z20 S by gradient sedimentation (our unpublished data). We have shown biochemical interactions between the Vam2/6p complex, the SNAREs, and Ypt7p, whereas the TRAPP and exocyst complexes have been shown to inter- act genetically with homologous factors (Bowser et al., 1992; Sacher et al., 1998). The Rab5p effector EEA1 can also interact with NSF and with syntaxin 6, and with com- plexes containing syntaxin 13 (McBride et al., 1999; Simon- sen et al., 1999), and thus there may be similarities to the functions of EEA1 and Vam2/6p. The yeast exocyst com- ponent Sec15p has also been shown to function with the plasma membrane Rab GTPase Sec4p (Guo et al., 1999), and Cao et al. (1998) have shown that the activity of Ypt1p is required for the proper localization of Uso1p to the cis- Golgi membrane in yeast. Like components of the TRAPP and exocyst complexes, Vam2p has homologues in other organisms, including worms, flies, plants, and mammals (Nakamura et al., 1997; Radisky et al., 1997; Warner et al., 1998). Significantly, the light gene product, the Drosophila VAM2 homologue, plays a role in protein trafficking to pigment granules, specialized lysosomes in the Drosophila eye, suggesting a conserved mode of action in lysosome biogenesis (Warner et al., 1998; Lloyd et al., 1998). We are only beginning to learn the full structural and functional complexity of the cis-SNARE complex. The core of this and other SNARE complexes is undoubtedly composed of a four-helix bundle (Katz et al., 1998; Poir- ier et al., 1998; Sutton et al., 1998) with a t-SNARE, v-SNARE, and s-SNARE. However, these core elements may be insufficient for function. The vacuole 65S complex contains the cis-SNARE complex, consisting of Vam3p, Vam7p, Nyv1p, Vti1p, Ykt6p, Sec17p, Sec18p, and LMA1, and Vam2/6p itself contains additional subunits (Seals, D., G. Eitzen, W. Wickner, and A. Price, manuscript in prepa- ration). Thus far, these multiple subunits do not appear to be redundant, as deletion or antibody inhibition of each of them (after priming, when the constituent subunits are separated from one another) has a major deleterious ef- fect on the reaction (Nichols et al., 1997; Ungermann and Wickner, 1998; Ungermann et al., 1999). We find a smaller percent of SNAREs in complex with Vam2/6p (Fig. 3 B) than the percent of Vam2/6p that is associated with SNAREs (Fig. 3 D), suggesting that there are extra cis- SNARE complexes without Vam2/6p. This fits with the observation that trans-SNARE complexes, which need Vam2/6p to form, only encompass a small percent of the SNAREs (Ungermann et al., 1998b), and with observa- tions in other systems such as yeast plasma membrane t-SNAREs being all over the bud rather than exclusively at the fusion site at the bud tip (Brennwald et al., 1994), and in neurons where t-SNAREs are not exclusively at the synapse (Garcia et al., 1995). Vam2/6p is localized in vivo to one or two discrete spots per vacuole (Nakamura et al., 1997), suggesting that like the exocyst on the plasma mem- brane, the localization of this complex determines the site of membrane docking. Critical testing of these ideas must await the determination of whether the same SNAREs that are in complex with Vam2/6p are those that form the trans-SNARE pairs. We thank Dr. Yoh Wada for providing the HA-VAM6 plasmid, Dr. Gary Eitzen for the pGST-Ypt7 construct, and Dr. Greg Payne for anti-Kex2p. We thank Drs. Charles Barlowe, Jerry Eichler, Gary Eitzen, Daniel Klionsky, Zuoyu Xu, and especially Dr. Tom Rapoport for helpful discus- sions, and Dr. Scott Emr for communicating unpublished results. We thank Li Wang for generously sharing preliminary results. This research was supported by a grant from the National Institute of General Medical Sciences. C. Ungermann was supported by a postdoc- toral fellowship from the Deutsche Forschungsgemeinschaft (DFG). Price et al. Yeast Vacuole Fusion 1237