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The Amino-terminal Domain of the E Subunit of Vacuolar H+-ATPase (V-ATPase) Interacts with the H Subunit and Is Required for V-ATPase Function

2002; Elsevier BV; Volume: 277; Issue: 41 Linguagem: Inglês

10.1074/jbc.m203521200

1083-351X

Ming Lu, Sandra Vergara, Li Zhang, L. Shannon Holliday, John P. Aris, Stephen L. Gluck,

Metalloenzymes and iron-sulfur proteins

Vacuolar H + -ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases transport protons by a rotary catalytic mechanism analogous to that used by F 1 F 0 -ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmed in vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominant-negative mutants resulted in dramatic decreases in bafilomycin A 1 -sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase.