Dec 1, 1995

Site-directed mutagenesis of the yeast PMA1 H(+)-ATPase. Structural and functional role of cysteine residues

The Journal of Biological Chemistry
V V Petrov, C W Slayman

Abstract

The yeast plasma-membrane H(+)-ATPase contains nine cysteines, three in presumed transmembrane segments (Cys-148, Cys-312, and Cys-867) and the rest in hydrophilic regions thought to be exposed at the cytoplasmic surface (Cys-221, Cys-376, Cys-409, Cys-472, Cys-532, and Cys-569). To gather new functional and structural information, we have studied the yeast ATPase by cysteine mutagenesis. It proved possible to replace seven of the nine cysteines by alanine, one at a time, without any significant decrease in ATP hydrolysis or ATP-dependent proton pumping. In the remaining two cases (Cys-409 and Cys-472), there were small but reproducible effects; the results clearly indicated, however, that no single Cys is required for activity and that, if a disulfide bridge is formed in the yeast ATPase, it does not play an obligatory structural or functional role. Next, multiple mutants were constructed to ask how many Cys residues could be replaced simultaneously while leaving a fully functional enzyme. After substitution of all "membrane" Cys (Cys-148, Cys-312, and Cys-867) together with two non-conserved Cys located in hydrophilic regions (Cys-221 and Cys-569), there were no significant abnormalities in expression (87%) or activity (89% A...Continue Reading

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Mentioned in this Paper

Saccharomyces cerevisiae Proteins
Adenosine Triphosphatases
Mutagenesis, Site-Directed
Integral to Membrane
Trypsin
Ligand Binding Domain
Cytoplasmic
Protein Conformation
Cysteine
PMA1 protein, S cerevisiae

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