Biochemical and functional characterization of the actin-binding activity of the B subunit of yeast vacuolar H+-ATPase.
Vacuolar H+-ATPase (V-ATPase) is a fundamentally important enzyme in eukaryotic cells that is responsible for acidification of endocytic compartments. The B subunits of V-ATPases from mammals and tobacco hornworm have been shown to bind actin filaments. Actin-binding activity by the B subunit is required for targeting V-ATPases to the plasma membrane of osteoclasts. Bacterially expressed B subunit from the yeast Saccharomyces cerevisiae bound actin filaments with a Kd of 195 nmol l(-1). The actin-binding domain of the B subunit was altered by mutations that reduced or eliminated the actin-binding activity. Mutants assembled properly with endogenous yeast subunits when expressed in B subunit-null yeast and bafilomycin-sensitive ATPase activity was not significantly different from yeast transformed with wild-type B subunit. Yeast containing the mutant subunits grew as well at pH 7.5 as wild-type. Screening null yeast or null yeast transformed with wild-type or mutant B subunits with sub-lethal doses of various drugs revealed that yeast containing the mutant B subunits were more sensitive to cycloheximide and wortmannin than those transformed with wild-type B subunits. These results suggest that actin-binding activity confers on the B subunit of yeast a function that is distinct from its role in the enzymatic activity of the proton pump.
Zuo
Jian
J
Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610, USA.
Vergara
Sandra
S
Kohno
Shinya
S
Holliday
L Shannon
LS
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D018942:Macrolides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D051304:Profilins / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D000077191:Wortmannin
D000199:Actins; D000730:Androstadienes; D018942:Macrolides; D051304:Profilins; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C040929:bafilomycin A1; D003513:Cycloheximide; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; D000077191:Wortmannin