title: Modification of the N-terminal polyserine cluster alters stability of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae.
abstract: The N-terminus of the H(+)-ATPase from Saccharomyces cerevisiae contains a serine-rich cluster of 11 serine residues in the first 17 amino acids, including a stretch of eight consecutive serine residues. This cluster is conserved in the weakly expressed PMA2 gene from the same organism, but it is not present in PMA genes from other organisms suggesting that it is not likely to represent a conserved functional motif. To better understand whether this region plays a regulatory role, a series of mutant enzymes were generated in which the serine tract was systematically converted to alanine or deleted. Conversion of the first six serine residues to alanine or deletion of the entire serine tract had little effect on cell growth phenotypes. However, when eight or more serines were converted, the mutant cells displayed prominent hygromycin B-resistant and low pH-sensitive phenotypes indicative of reduced H(+)-ATPase function. The mutant enzymes were found to display relatively normal kinetic properties for ATP hydrolysis, but showed significantly decreased abundance in the plasma membrane under stress conditions when eight or more serine residues were converted to alanine. The reduced abundance of the enzyme appeared to be due to degradative turnover, as mutant enzymes with multiple alanine substitutions showed an accelerated rate of turnover relative to wild-type. The polyserine tract in the H(+)-ATPase does not appear to be important for catalysis, but may contribute to overall protein stability.
authors.affiliation: Public Health Research Institute, 455 First Avenue, New York, NY 10016, USA.
authors.forename: A
authors.initials: A
authors.lastname: Hasper
authors.forename: P
authors.initials: P
authors.lastname: Soteropoulos
authors.forename: D S
authors.initials: DS
authors.lastname: Perlin
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D017931:DNA Primers; D010455:Peptides; C027794:polyserine; D006180:Proton-Translocating ATPases; D000409:Alanine