Mutagenesis analysis of the yeast Nha1 Na+/H+ antiporter carboxy-terminal tail reveals residues required for function in cell cycle.
The yeast Nha1 Na(+),K(+)/H(+) antiporter may play an important role in regulation of cell cycle, as high-copy expression of the NHA1 gene is able to rescue the blockage at the G(1)/S transition of cells lacking Sit4 protein phosphatase and Hal3 activities. Interestingly, this function was independent of the role of the antiporter in improving tolerance to sodium cations, it required the integrity of a relatively large region (from residues 800 to 948) of its carboxy-terminal moiety, and was not performed by the fission yeast homolog antiporter Sod2, which lacks a carboxy-terminal tail. Here we show that a hybrid protein composed of the Sod2 antiporter fused to the carboxy-terminal half of Nha1 strongly increased sodium tolerance, but did not allow growth at high potassium nor did rescue growth of the sit4 hal3 conditional mutant strain. Deletion of Nha1 residues from 800 to 849, 900 to 925 or 926 to 954 abolished the function of Nha1 in cell cycle without affecting sodium tolerance. A screening for loss-of-function mutations at the 775-980 carboxy-terminal tail of Nha1 has revealed a number of residues required for function in cell cycle, most of them clustering in two regions, from residues 869 to 876 (cluster A) and 918 to 927 (cluster B). The later is rather conserved in other related antiporters, while the former is not.
Simón
Ernesto
E
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain.
Barceló
Anna
A
Ariño
Joaquín
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002453:Cell Cycle; D017124:Conserved Sequence; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics* / Q000502:physiology
D000596:Amino Acids; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers