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.
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.
Lu
Ming
M
Department of Medicine University of Florida College of Medicine, Gainesville, Florida 32610, USA. luming@medicine.ufl.edu
Vergara
Sandra
S
Zhang
Li
L
Holliday
L Shannon
LS
Aris
John
J
Gluck
Stephen L
SL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D018942:Macrolides; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015003:Yeasts / Q000201:enzymology / Q000502:physiology
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D018942:Macrolides; D021122:Protein Subunits; D011522:Protons; D011993:Recombinant Fusion Proteins; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases