Modified plant plasma membrane H+-ATPase with improved transport coupling efficiency identified by mutant selection in yeast |
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Authors: | Lone Baunsgaard Kees Venema Kristian B Axelsen José Manuel Villalba Annikki Welling Bernd Wollenweber Michael G Palmgren |
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Institution: | Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark;Institute of Molecular Biology, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 Copenhagen, Denmark;Departamento de Biologia Celular, Universidad de Cordoba, Avda. San Alberto Magno, s/n, E-14004 Cordoba, Spain;Department of Agricultural Science, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark |
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Abstract: | 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 by 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 species 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 plants transporters. |
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