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 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.
Unité de biochimie physiologique, Université catholique de Louvain, Louvain-la-Neuve, Belgium.
P
P
Morsomme
A
A
de Kerchove d'Exaerde
S
S
De Meester
D
D
Thinès
A
A
Goffeau
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M
Boutry
D000255:Adenosine Triphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D010947:Plants, Toxic; D017354:Point Mutation; D017494:Proton Pumps / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000254:growth & development*; D014026:Nicotiana
D007527:Isoenzymes; D008244:Lysophosphatidylcholines; D017494:Proton Pumps; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D005947:Glucose