Regulation and pH-dependent expression of a bilaterally truncated yeast plasma membrane H+-ATPase.
Constitutive, chromosomal expression of yeast pma1 deletion alleles in Saccharomyces cerevisiae yielded functional, truncated forms of the plasma membrane H+-ATPase which were independently capable of supporting wild type yeast growth rates. Deletion of 27 amino-terminal residues affected neither the enzyme's activity nor its responsiveness to changes in glucose metabolism. By contrast, removal of 18 carboxy-terminal amino acids produced an enzyme with a Vmax that was relatively insensitive to glucose-dependent metabolic status and with a Km that was significantly lower than that of the wild type enzyme. These effects were exaggerated when the amino- and carboxy-terminal deletions were combined in a bilaterally truncated H+-ATPase, suggesting that the amino terminus may have a subtle role in modulating ATPase activity. In pma1DeltaDelta cells cultured at pH 6, plasma membrane H+-ATPase levels were much lower than those in cells expressing a wild type ATPase. Increased expression levels could be achieved by growing the pma1DeltaDelta mutant at pH 3, a result that was at least partially due to a sustained, elevated transcription of pma1DeltaDelta mRNA. Our observations suggest that intracellular proton balance can be maintained by regulation of the activity and/or quantity of H+-ATPase in the plasma membrane.
Mason
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AB
Molecular Microbiology Laboratory, Department of Oral Sciences, School of Dentistry and the Centre for Gene Research, University of Otago, P.O. Box 647, Dunedin, New Zealand.
Kardos
T B
TB
Monk
B C
BC
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; D012333:RNA, Messenger; D006180:Proton-Translocating ATPases; D005947:Glucose