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

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 PMA% 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.