Vacuolar H ϩ -ATPase (V-ATPase) 3 is a highly conserved ATP-driven proton pump distributed throughout the endomembrane system. Intracellular V-ATPase generates and maintains the acidic organelle luminal pH necessary for membrane trafficking, protein sorting and processing, and zymogen activation (1-5). V-ATPase is also present at the plasma membrane of cells specialized for active proton transport. Kidney intercalated cells (6), bone osteoclasts (7), and epididymis clear cells (6) express V-ATPases at the plasma membrane, which are necessary for systemic acid-base balance, bone resorption, and sperm maturation, respectively.V-ATPase consists of two multisubunit domains, V 1 and V o , that are responsible for ATP hydrolysis and proton transport, respectively (8, 9). Peripheral subunits form V 1 , the catalytic domain attached to the cytosolic side of the membrane. V 1 is bound to V o , the integral membrane domain that forms the path for proton transport. During catalysis, ATP hydrolysis within V 1 drives active transport of protons from the cytosol to the other side of the membrane via rotation of a proteolipid ring structure in V o . Detachment of V 1 from V o is an important mechanism that inhibits V-ATPase proton transport (3, 10, 11). Disassembly and reassembly of V 1 V o has been observed in yeast, insects, and mammalian cells (3,(11)(12)(13). However, the cellular mechanisms governing V-ATPase regulation by reversible disassembly are not well understood. In yeast, V-ATPase reversible disassembly is intertwined with cytosol pH changes (14, 15) the RAS/cAMP/PKA pathway (16), and glycolysis (3).Disassembly of the yeast V 1 V o complex occurs when glucose is not available. Upon glucose depletion, V 1 subunit C is released into the cytoplasm, causing the rest of the V 1 domain to dissociate from V o (10,17,18). Disassembly completely inactivates V-ATPase pumps because V 1 without V o cannot hydrolyze ATP, and V o without V 1 cannot transport protons (2). Consequently, protons cannot not be redistributed from the cytosol into the vacuole, which alkalinizes the vacuolar lumen and acidifies the cytosol (19,20). Disassembly is reversed by readdition of glucose to yeast cells (10,21). After reassembly, V 1 V o resumes ATP-driven proton transport, which restores vacuolar and cytosol pH homeostasis (20).Complete glucose depletion causes about 75% of the V 1 V o complexes to disassemble (10, 21); the remaining 25% of the pumps maintain the V-ATPase activity necessary to sustain basal cellular functions. Presumably yeast V 1 V o disassembly preserves energy when glucose, the main energy source, is lim-* This work was supported by National Institutes of Health Grant R01GM086495 (to K. J. P.) and American Heart Association Grant 14PRE19020015 (to C. Y. C.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Human THEM4 (hTHEM4) is comprised of a catalytically active hotdog-fold acyl-CoA thioesterase domain and an N-terminal domain of unknown fold and function. hTHEM4 has been linked to Akt1 regulation and cell apoptosis. Herein, we report the X-ray structure of hHTEM4 bound with undecan-2-one-CoA. Structure guided mutagenesis was carried out to confirm the catalytic residues. The N-terminal domain is shown to be partially comprised of irregular and flexible secondary structure, reminiscent of a protein-binding domain. We demonstrate direct hTHEM4-Akt1 binding by immunoprecipitation and by inhibition of Akt1 kinase activity, thus providing independent evidence that hTHEM4 is an Akt1 negative regulator.