The entire SGA protocol was repeated three times, and only candidates that were identified in two of the three independent screens are included in Table S1. and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p.STV1overexpression suppressed the growth defects of bothrav1andrav1vph1, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination withrav1perturbed the normal localization of Stv1green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vmaphenotypes arise from defects in Vph1p-containing complexes caused byrav1, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor. == INTRODUCTION == Vacuolar H+-translocating ATPases (V-ATPases) are highly conserved proton pumps responsible for acidification of organelles such as mammalian lysosomes, yeast or plant vacuoles, endosomes, Golgi apparatus, and regulated secretory granules in all eukaryotes (Kane, 2006;Forgac, 2007). V-ATPases are multisubunit complexes consisting of a subcomplex of peripheral membrane subunits (V1) attached to a complex of membrane subunits (Vo). Although higher eukaryotes often encode a number of different tissue- and organelle-specific isoforms, the yeastSaccharomyces cerevisiaecontains a single set of isoforms (Manolsonet al., 1994;Forgac, 2007).VPH1encodes the Vosubunitaisoform localized predominantly to the vacuole, andSTV1encodes the second isoform of Vosubunita, present in Golgi and endosomes (Manolsonet al., 1992;Kawasaki-Nishiet al., 2001a). Mechanistically, ATP hydrolysis in the V1sector is coupled to proton transport through the Vodomain via a rotational mechanism (Hirataet al., 2003). Detachment of V1sectors from Vosectors inactivates both subcomplexes, and AZD8797 serves as a mechanism for regulating V-ATPase activity. Disassembly of the V-ATPase is triggered rapidly by glucose deprivation, and reassembly also occurs rapidly upon glucose readdition (Kane, 2000). There is evidence that Stv1p-containing V-ATPases are less responsive to glucose control than Vph1p-containing complexes (Kawasaki-Nishiet al., 2001b). Regardless of subunitaisoform, V-ATPase complexes at the vacuole appear to undergo reversible disassembly more readily than V-ATPases in prevacuolar compartments (Kawasaki-Nishiet al., 2001b;Qi and Forgac, 2007). The regulator of ATPase of vacuoles and endosomes (RAVE) complex binds to the V1complex and subunit C in the cytosol and promotes their assembly with the membrane-bound Vocomplex (Seolet al., 2001;Smardonet al., 2002;Smardon and Kane, 2007). In addition to Skp1p, which is found in multiple complexes, including several ubiquitin ligases, the RAVE complex is composed of two other proteins, Rav1p and Rav2p (Seolet al., 2001). Biochemical approaches have revealed that Rav1p is the central component of the RAVE complex, with distinct binding sites for Rav2p, Skp1p, V1subunits E and/or G, and V1subunit C (Smardonet al., 2002;Smardon and Kane, 2007). Deletion of eitherRAV1orRAV2results in defective assembly of V-ATPase complexes at the AZD8797 vacuole, and both biosynthetic assembly of V-ATPases and reassembly AZD8797 of complexes disassembled in response to glucose deprivation were affected in therav1mutant (Seolet al., 2001;Smardonet al., 2002). Interestingly, the yeastrav1andrav2mutants both exhibit a partial Vmaphenotype, with the characteristic Vmagrowth defects at high pH and high Ca2+concentrations only observed at 37C. The source of this temperature MCH6 sensitivity is not well understood (Seolet al., 2001). Loss of RAVE function has also been reported to compromise transport between early endosomes and the prevacuolar compartment (Siposet al., 2004). RAV1homologues exist in higher eukaryotes as well, in which they are called rabconnectin-3 or DMX-like proteins (Kraemeret al., 1998;Naganoet al., 2002). Only fungi have homologues of yeastRAV2, but higher eukaryotes do have a second rabconnectin subunit (rabconnectin-3) that forms a complex with rabconnectin-3 and gives identical phenotypes to rabconnectin-3 when disrupted (Sakisaka and Takai, 2005;Yanet al., 2009). InDrosophilaand human cell lines, rabconnectin3- and rabconnectin3- have been implicated in regulation of organelle acidification and endosomal trafficking (Yanet al., 2009;Sethiet al., 2010). Loss of rabconnectin-3 function in these organisms is associated with Notch signaling defects that have been traced to a requirement for endosomal acidification in this signaling pathway (Yanet al., 2009;Sethiet al., 2010). In zebrafish, loss of rabconnectin function is associated with reduced acidification of synaptic vesicles.