Chimeric V‐ATPases with Different Regulatory Properties

Abstract

Organelles in the endocytic and secretory pathway are primarily acidified by V‐ATPase. Each of these organelles has a distinct luminal pH, but how this diverse pH range is established is not fully understood. V‐ATPases undergo different modes of regulation which may help fine tune their activity. The a‐subunit from the membrane bound Vo subcomplex is a regulatory hub of V‐ATPase and harbors many regulatory interactions in its cytosolic N‐terminal (aNT) domain. Vo a‐subunit exhibits organelle‐ and tissue‐specific isoforms. Vph1 and Stv1, the two organelle‐specific a‐subunit isoforms in yeast, reside in the lysosome‐like vacuole and Golgi apparatus, respectively. These isoforms differ in their dependence on the RAVE (Regulator of H+‐ATPase of vacuoles and endosomes) complex and phosphoinositide phospholipids (PIP lipids), two important factors previously implicated in isoform‐specific V‐ATPase regulation. We hypothesize that the aNT domain contains information for RAVE and PIP lipid dependent regulation. The aNT domains of all isoforms have a “dumbbell” shape with proximal and distal globular subdomains connected by a coiled‐coil. To better understand the regulatory information present in the aNT domain, we generated chimeras of Vph1 and Stv1 by swapping the proximal end and the distal ends between the isoforms. Vph1‐containing V‐ATPases require the RAVE complex for their assembly, but Stv1‐containing V‐ATPases assemble independent of RAVE. Stv1NT does not bind to RAVE subunits in vitro, but introduction of the proximal domain of Vph1NT fully restores the RAVE interaction, implicating this region of Vph1 in RAVE‐dependent assembly. The two isoforms also interact with distinct PIP lipids enriched in their organelle of residence; Stv1NT binds tightly to Golgi PI4P and Vph1NT binds to vacuolar PI(3,5)P2. Previous in vitro studies identified a PI4P binding site in the proximal domain of Stv1NT; a 6‐amino acid sequence containing this site proved to be sufficient to transfer PI4P binding to Vph1NT. However, lipid binding experiments with the chimeric aNTs indicate that both the proximal and the distal ends of Stv1NT contain sequences that promote PI4P binding. Previous studies implicated the Vph1NT distal domain in PI(3,5)P2‐dependent V‐ATPase regulation, but results with chimeric aNTs indicate that PI(3,5)P2 binds to the Vph1NT with low affinity and suggest the Vph1NT proximal domain may actually inhibit tight binding to PI(3,5)P2. Interestingly, when present as part of full‐length a‐subunit in yeast, 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 are fully functional there are consequences of their altered regulatory properties. V‐ATPases disassemble during glucose deprivation but do not reassemble efficiently after glucose re‐addition, consistent with lack of RAVE binding. Delayed growth in media containing raffinose suggests they cannot readily adjust during a transition to a less preferred carbon source. Together these data reveal the interplay between two mechanisms of V‐ATPase regulation and suggest that aNT domains can functionally integrate multiple regulatory inputs.