Structurally and Functionally Conserved Domains in the Diverse Hydrophilic Carboxy-Terminal Halves of Various Yeast and Fungal Na+/H+ Antiporters (Nhalp)

Genes encoding the Na+/H+antiporter (Nhalp) from Candida tropicalis (C.t.), Hansenula anomala (H.a.) (also named Pichia anomala), and Aspergillus nidulans (A.n.) were cloned, and the nucleotide sequences were determined. The deduced primary sequences revealed highly conserved hydrophobic regions and rather diverse hydrophilic regions. Among the seven known Nhalp sequences, Schizosaccharomyces pombe (S.p.) Nhalp is exceptional in lacking the hydrophilic region. Within the diverse hydrophilic regions, we found six conserved regions (C1-C6). Expression of C.t. Nhalp in Saccharomyces cer-evisiae (S.c.) cells lacking NHA1 and ENA1(Na+-ATPase) complemented the salinity-sensitive phenotype, suggesting that C.t. Nhalp is functionally related to S.c. Nhalp. Expression of various truncated forms of the C-terminal half of S.c. and C.t. Nhalp showed essentially the same phenotype for both species: deletion of the C4-C6 region caused cell growth to be more resistant to high salinity than the wild type, suggesting an inhibitory function of these domains on the antiporter activity. However, complete loss of C1-C6 caused a severe growth defect under conditions of high salinity, suggesting adefect in antiporter activity. The δC2-C6 form of C.t. Nhalp, containing only C1, restored the retarded cell growth at high salinity more than the control vector alone, but to a value lower than the wild type. These results suggest an essential role for C1 and an activating role of the C2-C3 region in the functional expression of Nhal. High expression of the δC2-C6 form of S.C. Nhalp was toxic for yeast cells, although low expression was not, suggesting that the overexpression of C1 is toxic. The results in this study suggest that the diverse hydrophilic region of yeast and fungal Nhalp has six conserved domains with conserved functions in terms of expression of Nhalp activity.