Isoform-Specific Regulation of the Yeast V-ATPase a-subunit

Abstract

V-ATPase primarily acidifies organelles of the endocytic and secretory pathway. The lumen of each of these organelles maintains a distinct pH, but how this diverse pH range is established is not well understood. Different modes of regulation of the V ATPase may help fine-tune their activity. The a-subunit from the membrane bound Vo subcomplex is a regulatory hub of V-ATPase which harbors many regulatory interactions in its cytosolic N-terminal (aNT) domain. The aNT domains of all isoforms consist of a proximal and a distal globular domain connected by a coiled-coil. Vo a-subunit exhibits organelle- and tissue-specific isoforms. Vph1 and Stv1 are the two organelle-specific a subunit isoforms in yeast. V-ATPase containing the Vph1 isoform reside in the lysosome-like yeast vacuole whereas V-ATPase containing the Stv1 isoform reside predominantly in the Golgi apparatus. The RAVE (Regulator of H+ -ATPase of vacuoles and endosomes) complex and phosphoinositide phospholipids (PIP lipids) are two important factors previously implicated in isoform-specific V-ATPase regulation. The a subunit isoforms vary in their dependence on the RAVE and PIP lipid. But where the information of RAVE and PIP lipid recognition reside in the aNT domain and how these two regulatory inputs are integrated to control V-ATPase function are not well understood. We hypothesize that the aNT domain contains distinct sequences for RAVE and PIP lipid recognition, and that the differential interactions of the a-subunit isoforms with RAVE and PIP lipids result in isoform-specific function and regulation of the V ATPase. To better understand the regulatory information present in the aNT domain, we generated chimeric aNT constructs by combining parts of Vph1NT and Stv1NT. Vph1NT was previously shown to bind to a RAVE subunit and Vph1 V-ATPases require the RAVE complex for their assembly. Stv1-containing V-ATPases on the other hand assemble independent of RAVE and Stv1NT does not bind to RAVE subunits in vitro. In chapter 2, we have shown that replacing the proximal domain of Vph1NT with the proximal end of Stv1NT fully restores the RAVE interaction, implicating this region of Vph1 in RAVE-dependent assembly. The two isoforms also exhibit preferences for distinct PIP lipids enriched in their organelle of residence; Stv1NT binds tightly to Golgi PI(4)P and Vph1NT binds to vacuolar PI(3,5)P2. A PI4P binding site in the proximal domain of Stv1NT was previously reported. Our analysis with chimeric constructs suggests that a 6-amino acid sequence containing this site is sufficient to transfer PI(4)P binding to Vph1NT. Our results also suggest that both the proximal and the distal ends of Stv1NT contain sequences that promote PI4P binding. Interestingly, when expressed in yeast as a full-length a-subunit, the chimera containing both PI4P and PI(3,5)P2 binding sites has wild-type level activity and assembly in isolated vacuoles even though it lacks a RAVE binding site. Although V-ATPases in this chimeric strain is fully functional there are consequences of their altered regulatory properties. V-ATPases in this chimera disassemble during glucose deprivation but do not reassemble efficiently after glucose re addition, consistent with a lack of RAVE binding. We also observed a delayed growth in media containing raffinose suggesting they cannot readily adjust during a transition to a less preferred carbon source. While the lack of RAVE binding gives rise to the growth defect of this chimera in a poor carbon source, increased PI(3,5)P2 binding of this mutant, on the other hand, contributes to growth benefit in alkaline pH stress. Together these data reveal the interplay between two mechanisms of V-ATPase regulation and suggest that aNT domains can functionally integrate multiple regulatory inputs.