The Docking Stage of Yeast Vacuole Fusion Requires the Transfer of Proteins from a cis-SNARE Complex to a Rab/Ypt Protein Albert Price, Darren Seals, William Wickner, and Christian Ungermann Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844 Abstract. The homotypic fusion of yeast vacuoles re- quires Sec18p (NSF)-driven priming to allow vacuole docking, but the mechanism that links priming and docking is unknown. We ﬁnd that a large multisubunit protein called the Vam2/6p complex is bound to cis- paired SNAP receptors (SNAREs) on isolated vacu- oles. This association of the Vam2/6p complex with the cis-SNARE complex is disrupted during priming. The Vam2/6p complex then binds to Ypt7p, a guanosine triphosphate binding protein of the Rab family, to ini- tiate productive contact between vacuoles. Thus, cis- SNARE complexes can contain Rab/Ypt effectors, and these effectors can be mobilized by NSF/Sec18p-driven priming, allowing their direct association with a Rab/Ypt protein to activate docking. Key words: Vps39/Vam6p • Vps41/Vam2p • Ypt7p • priming • Rab/Ypt effector Introduction Regulated fusion of organelle membranes (Rothman, 1994) is essential for subcellular protein compartmenta- tion. The protein and lipid factors that catalyze membrane fusion are being identified and their functional relation- ships are receiving intense study. Each fusion reaction seems to require integral membrane proteins termed SNAP receptors (SNAREs)1 (Pfeffer, 1996), which func- tion through the association of their cytoplasmic domains to form coiled-coils. Associated SNAREs, whether in cis (on the same membrane) or in trans (attached to apposed membranes), can be disassembled by the action of the ATP-driven chaperone, N-ethylmaleimide sensitive factor (NSF)/Sec18p, and its cochaperone aSNAP/Sec17p (Söll- ner et al., 1993; Hanson et al., 1995; Ungermann et al., 1998a). SNARE associations are regulated either directly or indirectly by members of the Rab/Ypt family of GTP- ases and their associated proteins, termed Rab/Ypt effec- tors (McBride et al., 1999; Carr et al., 1999; Ungermann Address correspondence to Bill Wickner, Department of Biochemis- try, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755- 3844. Tel.: (603) 650-1701. Fax: (603) 650-1353. A. Price’s present address is Department of Cell Biology, Sterling Hall of Medicine, C441, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208002, New Haven, CT 06520-8002. C. Ungermann’s present address is Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. The website for this laboratory is located at http://www.dartmouth.edu/ zwickner 1Abbreviations used in this paper: GST, glutathione S-transferase; HA, hemagglutinin; LMA1, low molecular weight activity #1; NSF, N-ethylma- leimide sensitive factor; SNARE, SNAP receptor; TRAPP, transport pro- tein particle. et al., 1998a). In several systems, Rab/Ypt proteins and their effectors have been shown to mediate a SNARE- independent initial association of membranes, termed teth- ering, which is the first step of the docking stage (Cao et al., 1998; Ungermann et al., 1998b). Tethering is a necessary prelude to trans-SNARE pairing, which establishes a sta- bly docked organelle pair. The recent finding (McBride et al., 1999) that Rab effectors can directly associate with SNAREs and that this association can be modulated by the chaperone NSF, suggested that these Rab effectors may directly mediate trans-SNARE pairing and the subse- quent steps that lead to fusion. We have studied the homotypic fusion of yeast vacuoles. This reaction occurs in obligately ordered subreactions of priming, docking, and fusion. Five SNAREs on the puri- fied organelle are found in a cis complex with the chaper- ones Sec18p, Sec17p, and low molecular weight activity #1 (LMA1) (Ungermann et al., 1999). This complex has been viewed as simply the remains of SNARE associations from prior fusion events, though current studies reported below show that it actually has a greater complexity and distinct functions. The cis-SNARE complex is disassembled as Sec18p hydrolyzes ATP during priming (Barnard et al., 1997; Ungermann et al., 1998b). This priming event is nec- essary for docking (Mayer and Wickner, 1997). Docking occurs in two stages, a reversible vacuole association termed tethering, which depends on the Rab-like GTPase Ypt7p, and the subsequent pairing of SNAREs in trans (Ungermann et al., 1998a). We report that Vam2/Vps41p and Vam6/Vps39p, identified in vacuole protein sorting (vps) screens for defective vacuole protein sorting (Stack ª The Rockefeller University Press, 0021-9525/2000/03/1231/8 $5.00 The Journal of Cell Biology, Volume 148, Number 6, March 20, 2000 1231–1238 http://www.jcb.org 1231