Organelle‐specific Regulation of V‐ATPase Activity by Inositol Phospholipids

Abstract

All eukaryotic cells have multiple acidic compartments, including lysosomes, endosomes, the Golgi apparatus, and regulated secretory granules, that are tuned to distinct pH ranges essential for their functions. It is not fully understood how precise organelle pH ranges are established and maintained in vivo. We have found that organelle‐specific isoforms of the yeast V‐ATPase, the proton pump primarily responsible for organelle acidification, specifically interact with phosphoinositides (PIs) characteristic of the organelles where they reside. V‐ATPases in the yeast lysosome‐like vacuole contain the Vph1 isoform of the Vo a‐subunit, the largest integral membrane subunit of the enzyme. The Vph1 isoform interacts specifically with the late endosome/vacuole‐specific lipid PI(3,5)P2, and V‐ATPase activity in the vacuole increases under conditions that trigger an increase in PI(3,5)P2, such as salt shock (Li et al (2014) Mol. Biol. Cell 25:1251). Recently we have found that the Golgi isoform of this subunit, Stv1, interacts specifically with PI(4)P, a PI lipid enriched in Golgi membranes. Both Stv1 and Vph1 contain N‐terminal cytosolic domains attached to an 8‐helix, C‐terminal transmembrane domain critical for proton transport. In yeast cells, an engineered cytosolic domain of Stv1 (Stv1NT) is recruited from the cytosol to the membrane in a PI(4)P‐dependent manner. In vitro, bacterially expressed Stv1NT binds specifically to PI(4)P‐containing liposomes in a flotation assay. Neither Vph1 nor Stv1 contain canonical PI binding sites, but we have identified a single point mutation in Stv1NT that compromises Stv1NT binding to PI(4)P‐containing liposomes. Introduction of this mutation into full‐length Stv1 in yeast cells has little effect on Stv1 localization, but reduces V‐ATPase function. This suggests that PI(4)P is required for activity of Stv1‐containing V‐ATPases at the Golgi apparatus. Consistent with this, a pik1ts mutation, which compromises PI(4)P synthesis at the Golgi, also compromises function of Stv1‐containing V‐ATPases. Taken together, these data suggest that isoforms of the V‐ATPase Vo a‐subunits interact with specific PI head groups, and that these interactions provide organelle‐specific input into V‐ATPase function. Mammalian cells express four isoforms of the Vo a‐subunit, corresponding to yeast Vph1/Stv1. In vitro experiments with expressed cytosolic domains of the human Vo a‐subunit isoforms reveal that some of these isoforms also interact with specific PI lipids. Specifically, the Vo a2 isoform, shown previously to function at the Golgi, binds specifically to PI(4)P. These data indicate that the extent of organelle acidification may be determined in part by resident Vo a‐subunit isoforms and their interactions with specific phosphoinositides, which provide both organelle identity and regulatory signals.Support or Funding InformationSupported by NIH GM50322