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.
A screening for multicopy suppressors of the G(1)/S blockage of a conditional sit4 hal3 mutant 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 Asp(266)-Asp(267) selectively abolished Na(+) efflux without modifying K(+) efflux and did not affect the capacity of Nha1 to relieve the G(1) 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.
Simón
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E
Departament de Bioquimica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain.
Clotet
J
J
Calero
F
F
Ramos
J
J
Ariño
J
J
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001692:Biological Transport; D002176:Candida albicans / Q000378:metabolism; D027682:Cation Transport Proteins; D002412:Cations; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics*; D008565:Membrane Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D054648:Protein Phosphatase 2; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology*; D013489:Suppression, Genetic; D013997:Time Factors
D027682:Cation Transport Proteins; D002412:Cations; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; C000631624:NHA1 protein, S cerevisiae; C092936:SIS2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017923:Sodium-Hydrogen Exchangers; D001224:Aspartic Acid; D012964:Sodium; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C104343:SIT4 protein, S cerevisiae; D011188:Potassium