Epitope-tagged constructs of the yeast plasma-membrane H(+)-ATPase.
In this study, two different epitope tags (HA, c-myc) were introduced near the N terminus of the yeast PMA1 H(+)-ATPase. The resulting proteins were indistinguishable from the wild-type ATPase in their ability to travel through the secretory pathway, as judged by quantitative immunoblotting of isolated secretory vesicles. Furthermore, there were no significant abnormalities in ATPase activity (including K(m) for MgATP, Vmax, pH optimum, and IC50 for inhibition by vanadate) or in ATP-dependent proton pumping. Finally, the epitope-tagged ATPases could support normal growth and displayed the expected activation by glucose.
dos Santos
C F
CF
Department of Genetics, Yale University School of Medicine, New Haven, CT 06517, USA. tourinho@unisul.rct_sc.br
DeWitt
N D
ND
Gupta
S S
SS
Allen
K E
KE
Slayman
C W
CW
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D000939:Epitopes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D000939:Epitopes; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases