The Candida albicans Na(+)/H(+) antiporter exports potassium and rubidium.

The Candida albicans Cnh1p belongs to the family of Na(+)/H(+) antiporters (TC 2.A.36) but it transports besides toxic sodium and lithium also rubidium and potassium. Upon heterologous expression in a Saccharomyces cerevisiae salt-sensitive strain, the Cnh1p is targeted to the plasma membrane and its transport activity results in increased tolerance of cells to external alkali metal cations. The cation efflux activity of Cnh1p in S. cerevisiae depends on the gradient of protons across the plasma membrane, and a Cnh1p-mediated K(+) efflux is involved in a cell response to sudden rise of cytoplasmic pH.

The Zygosaccharomyces rouxii strain CBS732 contains only one copy of the HOG1 and the SOD2 genes.

The osmotolerant yeast Zygosaccharomyces rouxii CBS732 contains only one copy of the ZrHOG1 and ZrSOD2-22 genes. Both genes were cloned and sequenced (Acc. Nos. AJ132606 and AJ252273, respectively) and their sequences were compared to homologous pairs of genes from Z. rouxii ATCC42981 (genes Z-HOG1, Z-HOG2, Z-SOD2, Z-SOD22). The CBS732 ZrHog1p is shorter than its ATCC42981 counterparts (380 aa residues vs. 407 and 420 aa, respectively) and is more similar to ATCC42981 Z-Hog2p than to Z-Hog1p. Also its promoter region corresponds to that one of Z-HOG2. The CBS732 ZrHOG1 promoter region is recognised by Saccharomyces cerevisiae, and the gene product (MAP kinase ZrHog1p) presence fully complements the osmosensitivity of a S. cerevisiae hog1 mutant strain. The CBS ZrSOD2-22 gene is highly similar to ATCC42981 Z-SOD2 but it contains also a segment of 15 aa residues specific for Z-SOD22. Z. rouxii ZrSod2-22 Na(+)/H(+) antiporter expressed in S. cerevisiae shows better activity toward toxic Na(+) and Li(+) cations than does S. cerevisiae's own Nha1 antiporter, and is efficient in improving the halotolerance of some S. cerevisiae wild types.

Functional study of the Saccharomyces cerevisiae Nha1p C-terminus.

Saccharomyces cerevisiae cells possess an alkali metal cation antiporter encoded by the NHA1 gene. Nha1p is unique in the family of yeast Na+/H+ antiporters on account of its broad substrate specificity (Na+, Li+, K+) and its long C-terminus (56% of the whole protein). In order to study the role of the C-terminus in Nha1p function, we constructed a series of 13 truncated NHA1 versions ranging from the complete one (2958 nucleotides, 985 amino acids) down to the shortest version (1416 nucleotides, 472 amino acids), with only 41 amino acid residues after the last putative transmembrane domain. Truncated NHA1 versions were expressed in an S. cerevisiae alkali metal cation-sensitive strain (B31; ena1-4Delta nha1Delta). We found that the entire Nha1p C-terminus domain is not necessary for either the proper localization of the antiporter in the plasma membrane or the transport of all four substrates (we identified rubidium as the fourth Nha1p substrate). Partial truncation of the C-terminus of about 70 terminal amino acids improves the tolerance of cells to Na+, Li+ and Rb+ compared with cells expressing the complete Nha1p. The presence of the neighbouring part of the C-terminus (amino acids 883-928), rich in aspartate and glutamate residues, is necessary for the maintenance of maximum Nha1p activity towards sodium and lithium. In the case of potassium, the participation of the long C-terminus in the regulation of intracellular potassium content is demonstrated. We also present evidence that the Nha1p C-terminus is involved in the cell response to sudden changes in environmental osmolarity.

Degradation of Candida albicans Can1 permease expressed in Saccharomyces cerevisiae.

The Candida albicans amino-acid Can1 permease expressed in Saccharomyces cerevisiae is degraded in the vacuole after internalisation by endocytosis. The CaCan1 inactivation and degradation is slow and not inducible by ammonium ions or 'stress' conditions. Using Saccharomyces cerevisiae mutants defective in ubiquitin-protein ligase and ubiquitin-protein hydrolase we have shown that the degradation of heterologous CaCan1 permease is ubiquitin dependent.