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

EMBO J. 1996 Oct 15;15(20):5513-26.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Cell Membrane / enzymology
  • Glucose / metabolism
  • Hydrogen-Ion Concentration
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Kinetics
  • Lysophosphatidylcholines / pharmacology
  • Plants, Toxic
  • Point Mutation*
  • Proton Pumps / metabolism*
  • Proton-Translocating ATPases / genetics
  • Proton-Translocating ATPases / metabolism*
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / growth & development*
  • Tobacco

Substances

  • Isoenzymes
  • Lysophosphatidylcholines
  • Proton Pumps
  • Adenosine Triphosphate
  • Proton-Translocating ATPases
  • Glucose