Interactions between the aNT Domains of Human V‐ATPases and Phosphatidylinositol Phosphate Lipids

Abstract

Each organelle in a eukaryotic cell has a tightly regulated pH important for organelle function and cell growth. This distinct pH defines organelle identity and is maintained principally by vacuolar H+‐ATPases (V‐ATPases). V‐ATPases are highly conserved, ATP driven proton pumps comprised of a cytosolic V1 domain, and an integral membrane bound Vo domain. The cytosolic N‐terminal domain of the Vo a‐subunit is known to modulate the organelle specific regulation and targeting of V‐ATPases. However, the mechanisms for targeting V‐ATPases to distinct membranes and achieving organelle‐specific regulation are incompletely understood. Importantly, loss of function in V‐ATPase a‐subunit isoforms is associated with human diseases such as osteopetrosis, distal renal tubule acidosis, and cutis laxa type‐II. Organelles also have characteristic phosphatidylinositol phosphate (PIP) lipids in the outer leaflet of their membranes. Previous studies have demonstrated that the N‐terminal domain of yeast Vo a‐subunit isoforms, Vph1NT and Stv1NT, interact with distinct PIP lipids in their resident organelle and can affect activity, regulation, and localization of V‐ATPases containing these isoforms. We hypothesize that V‐ATPases and PIP lipids interact with the NT domains of human Vo a‐subunit isoforms, and these interactions regulate activity and targeting of V‐ATPase, thereby impacting pH‐dependent functions of the organelle. The Hua1 isoform resides in lysosomes of many cells and in synaptic vesicles of neurons, and the Hua2 isoform functions in Golgi and endosomes of multiple cell types. We have expressed the N‐terminal domains of human Vo a‐subunit isoforms Hua1NT and Hua2NT in E.coli, then purified and tested their specificity for different PIP lipids in a liposome pelleting assay. Hua1NT shows a preference for liposomes containing PI(3)P or PI(3,5)P2, lipids typically enriched in endosomes and lysosomes, while Hua2NT shows a preference for Golgi‐enriched lipid PI4P. Modeling on existing structures has identified potential PIP binding sites in the HuaNT domains, which were mutagenized and tested for PIP lipid specificity. We have identified PIP‐specific binding sites for both Hua1NT and Hua2NT. We will assess PIP‐specific membrane recruitment of wild‐type and mutant HuaNTs by monitoring their localization when transiently expressed in mammalian cells. Together, these data show that the association between V‐ATPase subunit isoforms and PIPs is preserved in mammalian cells. Defining PIP binding codes on V‐ATPase will improve our understanding of organelle specific pH control and could provide new avenues for controlling V‐ATPase subpopulations.