Modified plant plasma membrane H(+)-ATPase with improved transport coupling efficiency identified by mutant selection in yeast.
Transport across the plasma membrane is driven by an electrochemical gradient of H+ ions generated by the plasma membrane proton pump (H(+)-ATPase). Random mutants of Arabidopsis H(+)-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H(+)-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced gy site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200-500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H+ extrusion by transformed yeast cells. The different specifies of aha1, however, displayed marked differences in initial rates of net H+ extrusion and in their ability to sustain an electrochemical H+ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H(+)-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H+ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plant transporters.
Baunsgaard
L
L
Department of Plant Biology, Royal Veterinary and Agricultural University, Copenhagen, Denmark.
Venema
K
K
Axelsen
K B
KB
Villalba
J M
JM
Welling
A
A
Wollenweber
B
B
Palmgren
M G
MG
D000255:Adenosine Triphosphate / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017494:Proton Pumps / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic; D014364:Tryptophan / Q000502:physiology
C434364:PMA2 protein, S cerevisiae; D017494:Proton Pumps; D029701:Saccharomyces cerevisiae Proteins; D014364:Tryptophan; D000255:Adenosine Triphosphate; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases