Functional analysis of chimerical plasma membrane H+-ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe.
The plasma membrane H+-ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H+-ATPase-depleted cells of the budding yeast Saccharomyces cerevisiae, even after deletion of the enzyme's carboxy terminus. Functional chimerical H+-ATPase proteins in which appropriate regions of the S. pombe enzyme were replaced with their S. cerevisiae counterparts were generated by in vivo gene recombination. Site-directed mutagenesis of the H+-ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S. pombe H+-ATPase. The reverse Ala-598-->Tyr substitution was introduced into the S. cerevisiae enzyme to better understand the role of this alanine residue. However, no obvious effect on ATPase activity could be detected. The S. cerevisiae cells expressing the S. pombe H+-ATPase substituted with alanine were enlarged and grew more slowly than wild-type cells. ATPase activity showed a more alkaline pH optimum, lower K(m) values for MgATP and decreased Vmax compared with wild-type S. cerevisiae activity. None of these kinetic parameters was found to be modified in glucose-starved cells, indicating that the S. pombe H+-ATPase remained fully active. Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S. cerevisiae sequence spans most of the catalytic site.
de Kerchove d'Exaerde
A
A
Unité de Biochimie Physiologique, Université catholique de Louvain, Louvain-la-Neuve, Belgium.
Morsomme
P
P
Sempoux-Thinès
D
D
Supply
P
P
Goffeau
A
A
Ghislain
M
M
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012568:Schizosaccharomyces / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D006180:Proton-Translocating ATPases