A Screening for High Copy Suppressors of the sit4 hal3 Synthetically Lethal Phenotype Reveals a Role for the Yeast Nha1 Antiporter in Cell Cycle Regulation

• Published in: The Journal of biological chemistry 2001-08, Vol.276 (32), p.29740-29747
• Main Authors: Simón, Ernesto, Clotet, Josep, Calero, Fernando, Ramos, José, Ariño, Joaquı́n
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Aspartic Acid - chemistry; Biological Transport; Candida albicans; Candida albicans - metabolism; Cation Transport Proteins; Cations; Cell Cycle; Cell Cycle Proteins; DNA Mutational Analysis; Ena1 protein; Escherichia coli - metabolism; Flow Cytometry; Fungal Proteins - genetics; HAL3 gene; Membrane Proteins - physiology; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Na+,K+/H+ antiporter; Na+/H+-exchanging ATPase; Na@u+,K@u+/H@u+ antiporter; Na@u+/H@u+-exchanging ATPase; NHA1 gene; Phenotype; Phosphoprotein Phosphatases - genetics; Plasmids - metabolism; Potassium - chemistry; Potassium - metabolism; Protein Phosphatase 2; Protein Structure, Tertiary; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe; Sequence Homology, Amino Acid; SIT4 gene; Sod2 protein; Sodium - metabolism; Sodium - pharmacology; Sodium-Hydrogen Exchangers - physiology; Suppression, Genetic; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M101992200

A screening for multicopy suppressors of the G1/S blockage of a conditional sit4 hal3mutant yielded the NHA1 gene, encoding a Na+,K+/H+ antiporter, composed of a transmembrane domain and a large carboxyl-terminal tail, which has been related to cation detoxification processes. Expression of either the powerful Saccharomyces cerevisiae Ena1 Na+/H+-ATPase or the Schizosaccharomyces pombe Sod2 Na+/H+ antiporter, although increasing tolerance to sodium, was unable to mimic the Nha1 function in the cell cycle. Mutation of the conserved Asp residues Asp266-Asp267 selectively abolished Na+ efflux without modifying K+ efflux and did not affect the capacity of Nha1 to relieve the G1 blockage. Mutagenesis analysis revealed that the region near the carboxyl-terminal end of Nha1 comprising residues 800–948 is dispensable for sodium detoxification but necessary for transport of K+ cations. Therefore, this portion of the protein contains structural elements that selectively modulate Nha1 antiporter functions. This region is also required for Nha1 to function in the cell cycle. However, expression of the closely related Cnh1 antiporter from Candida albicans, which also contains a long carboxyl-terminal extension, although allowing efficient K+transport does not relieve cell cycle blockage. This indicates that although the determinants for Nha1-mediated regulation of potassium transport and the cell cycle map very closely in the protein, most probably the function of Nha1 on cell cycle is independent of its ability to extrude potassium cations.