MBoC E13-05-0231 revised The RAVE complex is an isoform-specific V-ATPase assembly factor in yeast

Abbreviations: RAVE, regulator of ATPase of vacuoles and endosomes; V-ATPase, vacuolar H-ATPase; SGA, synthetic genetic analysis; BCECF-AM, 2',7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, acetoxymethyl ester; YEPD, yeast extract-peptone-dextrose medium; SC, fully supplemented minimal medium ABSTRACT The RAVE complex is implicated in V-ATPase assembly and activity. In yeast, rav1∆ mutants exhibit a Vma growth phenotype, characteristic of loss of V-ATPase activity, only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperatureindependent Vma growth defect when combined with the rav1∆ mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1∆ mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the Nterminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1∆ and rav1∆ vph1∆, and allowed RAVE-independent assembly of active Stv1p-containing VATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1∆ perturbed the normal localization of Stv1-GFP. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma phenotypes arise from defects in Vph1p-containing complexes caused by rav1∆, 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 tissueand organelle-specific isoforms, the yeast S. cerevisiae contains a single set of isoforms (Manolson et al., 1994; Forgac, 2007). VPH1 encodes the Vo subunit a isoform localized predominantly to the vacuole, and STV1