Functional complementation of a null mutation of the yeast Saccharomyces cerevisiae plasma membrane H(+)-ATPase by a plant H(+)-ATPase gene.
In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The presence within an organ of several isoforms prevents a detailed enzymatic characterization of individual H(+)-ATPases. We therefore used the yeast Saccharomyces cerevisiae as a heterologous host for the expression of PMA2, an H(+)-ATPase isoform of Nicotiana plumbaginifolia. Yeast transformed by the plant pma2 was still able to grow under conditions where the yeast ATPase gene (PMA1) was either repressed or deleted. The transformed yeast strain was resistant to hygromycin, and its growth was prevented when the medium pH was lowered to 5.0. The N. plumbaginifolia PMA2 expressed in S. cerevisiae has unusual low Km for ATP (23 microM) and high pH optimum (6.8). Electron microscopic examination revealed PMA2 in internal structures of the karmellae type which proliferated when cell growth was arrested, either at a nonpermissive pH or at the stationary phase in a minimal medium. Under the latter conditions, subcellular fractionation on sucrose gradients revealed, in addition to the expected plant PMA2 peak linked to the plasma membrane fraction, low density peak containing PMA2 and KAR2, an endoplasmic reticulum marker. These observations suggest that the partial internal accumulation of PMA2 occurs in membranes derived from the endoplasmic reticulum and largely depends on growth conditions.
de Kerchove d'Exaerde
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Unité de Biochimie Physiologique, Université Catholique de Louvain, Belgium.
Supply
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Dufour
J P
JP
Bogaerts
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P
Thinés
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D
Goffeau
A
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Boutry
M
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D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005656:Fungal Proteins; D015870:Gene Expression; D005800:Genes, Fungal; D017343:Genes, Plant; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics; D010947:Plants, Toxic; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013347:Subcellular Fractions / Q000201:enzymology; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics*; D014170:Transformation, Genetic
D005656:Fungal Proteins; D007527:Isoenzymes; C434364:PMA2 protein, S cerevisiae; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases