Dissecting Regulatory Interactions with Cytosolic N‐terminal Domain of V‐ATPase a‐subunit Isoforms

Abstract

V-ATPases acidify lysosomes, endosomes, Golgi apparatus, and regulated secretory granules. Each of these organelles has a distinct luminal pH, but it is not fully understood how this pH is established or how V-ATPase subpopulations respond specifically to extracellular stimuli. The V-ATPase a-subunit of the integral membrane Vo subcomplex occupies a critical position in the enzyme and is a major focus of regulatory interactions. The Vo a-subunit is present as organelle- and tissue-specific isoforms in most organisms. All isoforms consist of a cytosolic N-terminal domain (aNT) and an integral membrane C-terminal domain. The aNT domains have a “dumbbell” shape with proximal and distal globular subdomains connected by a coiled-coil. We are dissecting the role of aNT in isoform-specific V-ATPase regulation. The two organelle-specific a-subunit isoforms in yeast, Vph1 and Stv1, reside in the lysosome-like vacuole and Golgi apparatus, respectively. These isoforms differ in their dependence on the RAVE (regulator of ATPase of vacuoles and endosomes) complex. Vph1-containing V-ATPases require the RAVE complex for their assembly, but Stv1-containing V-ATPases assemble stably in the absence of RAVE. We have generated chimeras of Vph1NT and Stv1NT, using information from recent structural studies. 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 phosphoinositide phospholipids enriched in their organelle of residence; Stv1NT binds tightly to Golgi PI4P and Vph1NT binds to vacuolar PI(3,5)P2. Mutational data identified a PI4P binding site in the proximal domain of Stv1NT, and chimeras of Stv1NT and Vph1NT support this is as the major binding site. Mutations in the Vph1NT distal domain disrupt PI(3,5)P2-dependent V-ATPase regulation, but results with chimeras indicate that PI(3,5)P2 binding to the Vph1NT distal domain is much weaker than PI4P binding to Stv1NT. The human genome encodes four a-subunit genes that also exhibit isoform-specific regulation. The a2 isoform is enriched in the Golgi/endosomes and the a1 isoform resides in endosomes and lysosomes. The a2NT domain binds specifically to PI4P-containing liposomes in vitro and the a1NT domain interacts with PI3P and PI(3,5)P2, two lipids enriched in endosomes and lysosomes. These data indicate that Vo a-subunit isoforms contain an “isoform code” that imparts V-ATPase subpopulations with distinct properties.