Structural and functional separation of the N- and C-terminal domains of the yeast V-ATPase subunit H.
The H subunit of the yeast V-ATPase is an extended structure with two relatively independent domains, an N-terminal domain consisting of amino acids 1-348 and a C-terminal domain consisting of amino acids 352-478. We have expressed these two domains independently and together in a yeast strain lacking the H subunit (vma13Delta mutant). The N-terminal domain partially complements the growth defects of the mutant and supports approximately 25% of the wild-type Mg(2+)-dependent ATPase activity in isolated vacuolar vesicles, but surprisingly, this activity is both largely concanamycin-insensitive and uncoupled from proton transport. The C-terminal domain does not complement the growth defects, and supports no ATP hydrolysis or proton transport, even though it is recruited to the vacuolar membrane. Expression of both domains in a vma13Delta strain gives better complementation than either fragment alone and results in higher concanamycin-sensitive ATPase activity and ATP-driven proton pumping than the N-terminal domain alone. Thus, the two domains make complementary contributions to structural and functional coupling of the peripheral V(1) and membrane V(o) sectors of the V-ATPase, but this coupling does not require that they be joined covalently. The N-terminal domain alone is sufficient for activation of ATP hydrolysis in V(1), but the C-terminal domain is essential for proper communication between the V(1) and V(o) sectors.
Liu
Mali
M
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
Tarsio
Maureen
M
Charsky
Colleen M H
CM
Kane
Patricia M
PM
D000255:Adenosine Triphosphate / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017136:Ion Transport; D018942:Macrolides / Q000494:pharmacology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017494:Proton Pumps; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
D004791:Enzyme Inhibitors; D018942:Macrolides; D017494:Proton Pumps; D011522:Protons; C036978:concanamycin A; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases