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. 1996 Oct 15;15(20):5513–5526.

Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

P Morsomme 1, A de Kerchove d'Exaerde 1, S De Meester 1, D Thinès 1, A Goffeau 1, M Boutry 1
PMCID: PMC452296  PMID: 8896445

Abstract

In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway.

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