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Shen, J, Hsu, CM, Kang, BK, Rosen, BP and Bhattacharjee, H (2003) The Saccharomyces cerevisiae Arr4p is involved in metal and heat tolerance. Biometals 16:369-78
Homologues of the bacterial ArsA ATPase are found in nearly every organism. While the enzyme is involved in arsenic detoxification in bacteria, the roles of eukaryotic homologues have not been identified. This article reports the function of the Saccharomyces cerevisiae homologue encoded by ARR4 gene (YDL100c ORF). Disruption of ARR4 was not lethal, but the disrupted strain displayed increased sensitivity to As3+, As5+, Co2+, Cr3+, Cu2+ or VO4(3-) salts and temperature. A plasmid-encoded copy of a wild-type ARR4 gene could complement the heat- or metal-related stress responses. Mutation of a codon within the consensus sequence for the nucleotide-binding site resulted in loss of complementation of the disrupted strain and produced a dominant negative phenotype in a wild type strain. Wild type and mutant Arr4p were purified from Escherichia coli. The wild type protein exhibited a low level of ATPase activity, and the mutant was inactive. The purified ATPase eluted as a dimer of 80-kDa species. A fusion of ARR4 and the GFP (green fluorescent protein) gene was constructed. The gene fusion was able to complement stress-related phenotype of the ARR4 disruption. Under non-stress conditions, GFP fluorescence was found diffusely in the cytosol. Under stress conditions GFP was localized in a few punctate bodies resembling late endosomes. It is proposed that under heat or metal stress, the soluble ATPase becomes membrane-associated, perhaps through interaction with a partner protein, and that this complex is involved in stress tolerance.
Adaptation, Physiological/genetics; Adenosine Triphosphatases; Arsenate Reductases; Arsenite Transporting ATPases; Arsenites/pharmacology; Cell Division/drug effects; Cell Division/genetics; Dose-Response Relationship, Drug; Drug Resistance, Microbial/genetics; Green Fluorescent Proteins; Guanine Nucleotide Exchange Factors/genetics; Guanine Nucleotide Exchange Factors/physiology; Ion Pumps/genetics; Ion Pumps/physiology; Luminescent Proteins/genetics; Luminescent Proteins/metabolism; Metals, Heavy/toxicity; Microscopy, Confocal; Molecular Weight; Multienzyme Complexes/genetics; Multienzyme Complexes/physiology; Mutation; Phenotype; Saccharomyces cerevisiae/drug effects; Saccharomyces cerevisiae/enzymology; Saccharomyces cerevisiae/genetics; Saccharomyces cerevisiae Proteins/genetics; Saccharomyces cerevisiae Proteins/physiology; Sodium Compounds/pharmacology; Temperature; Time Factors
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