Identification of a domain in the V0 subunit d that is critical for coupling of the yeast vacuolar proton-translocating ATPase.
Vacuolar proton-translocating ATPase pumps consist of two domains, V(1) and V(o). Subunit d is a component of V(o) located in a central stalk that rotates during catalysis. By generating mutations, we showed that subunit d couples ATP hydrolysis and proton transport. The mutation F94A strongly uncoupled the enzyme, preventing proton transport but not ATPase activity. C-terminal mutations changed coupling as well; ATPase activity was decreased by 59-72%, whereas proton transport was not measurable (E328A) or was moderately reduced (E317A and C329A). Except for W325A, which had low levels of V(1)V(o), mutations allowed wild-type assembly regardless of the fact that subunits E and d were reduced at the membrane. N- and C-terminal deletions of various lengths were inhibitory and gradually destabilized subunit d, limiting V(1)V(o) formation. Both N and C terminus were required for V(o) assembly. The N-terminal truncation 2-19Delta prevented V(1)V(o) formation, although subunit d was available. The C terminus was required for retention of subunits E and d at the membrane. In addition, the C terminus of its bacterial homolog (subunit C from T. thermophilus) stabilized the yeast subunit d mutant 310-345Delta and allowed assembly of the rotor structure with subunits A and B. Structural features conserved between bacterial and eukaryotic subunit d and the significance of domain 3 for vacuolar proton-translocating ATPase function are discussed.
Owegi
Margaret A
MA
Department of Chemistry, Ball State University, Muncie, Indiana 47306, USA.
Pappas
Donald L
DL
Finch
Mark W
MW
Bilbo
Sarah A
SA
Resendiz
Cruz A
CA
Jacquemin
Lori J
LJ
Warrier
Aswathy
A
Trombley
John D
JD
McCulloch
Kathryn M
KM
Margalef
Katrina L M
KL
Mertz
Melissa J
MJ
Storms
Jason M
JM
Damin
Craig A
CA
Parra
Karlett J
KJ
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C091579:VMA8 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases