Phosphoinositide-Specific binding by Human V-ATPase a-subunit isoforms
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Term and YearSpring 2023
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AbstractThe individual organelles in a eukaryotic cell have tightly regulated pH, essential for their function and viability. 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 peripheral, cytosolic V1 domain, and an integral membrane bound Vo domain. The cytosolic N-terminal domain of the Vo a-subunit (aNT), positioned at the interface of V1 and Vo, modulates organelle specific regulation and targeting of V-ATPases. The a-subunit is encoded by several tissue and organelle-specific isoforms that help target V-ATPases to various organelles and confer distinct functional properties. Importantly, loss of V-ATPase a-subunit isoform function is associated with human diseases, making V-ATPases potential drug targets. However, the mechanisms for targeting V-ATPases to distinct membranes and achieving organelle-specific regulation are incompletely understood. Phosphatidylinositol phosphates (PIP) are low abundance lipids localized in the outer leaflets of organelle membranes and implicated in V-ATPase regulation and organelle pH maintenance. Studies have shown that the yeast a-subunit isoforms, Vph1NT and Stv1NT, interact with distinct PIPs in their resident organelle and affect activity, regulation, and localization of V-ATPases accommodating these isoforms. Higher organisms, including humans express four a-subunit isoforms. We hypothesize that V-ATPases and PIP lipids interact with the NT domains of human Vo a-subunit isoforms. The Hua1 and Hua2 isoforms function in endolysosomes and Golgi respectively. Our data shows that bacterially expressed HuaNTs bind specific PIP lipids, Hua1NT binds endosome/lysosome enriched PI(3)P and PI(3,5)P2 and Hua2NT bind Golgi-enriched PI(4)P. Cryo-EM structures from yeast and mammals show that aNT is dumbbell shaped, with globular proximal and distal ends supporting specific interactions with V1 and Vo subunits with poorly conserved loops facing the membrane. Modeling on existing structures has identified potential PIP binding sites in the HuaNT domains, which were mutagenized and tested for PIP specificity. In both the isoforms, binding sites were identified in the distal domain loops, highlighting their importance in PIP specificity of the a-subunit. Defining PIP binding codes on V-ATPase will improve our understanding of organelle specific pH control and provide new avenues for controlling V-ATPase subpopulations.
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