Modified plant plasma membrane H(+)-ATPase with improved transport coupling efficiency identified by mutant selection in yeast.
Plant J
; 10(3): 451-8, 1996 Sep.
Article
en En
| MEDLINE
| ID: mdl-8811859
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 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.
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Banco de datos:
MEDLINE
Asunto principal:
Saccharomyces cerevisiae
/
Arabidopsis
/
ATPasas de Translocación de Protón
/
Proteínas de Saccharomyces cerevisiae
/
Mutación
Idioma:
En
Año:
1996
Tipo del documento:
Article