RESUMO
The KHA1 gene from Debaryomyces hansenii has been identified and characterized by heterologous expression in Saccharomyces cerevisiae. The gene is orthologous to ScKHA1, previously reported in S. cerevisiae, and on the basis of the deduced amino acid sequence, DhKha1p can be classified as an Na(+)/H(+) transporter. Reverse transcriptase (RT)-PCR experiments indicated that the expression level of DhKHA1 was not dependent on high pH or on the presence of a high salt level in the growth medium. Overexpression of DhKHA1 in a salt-sensitive S. cerevisiae mutant (ena1-4 nha1 kha1) rendered cells specifically more tolerant to Na(+). In addition, internal K(+) and Na(+) measurements and experiments performed with green fluorescence protein (GFP)-tagged DhKha1p indicated the intracellular localization of this protein when expressed in S. cerevisiae.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Saccharomycetales/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Clonagem Molecular , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Dados de Sequência Molecular , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Trocadores de Sódio-Hidrogênio/genética , Fatores de TempoRESUMO
Debaryomyces hansenii is a salt-tolerant yeast that contains high amounts of internal Na(+). Debaryomyces hansenii kept more sodium than Saccharomyces cerevisiae in both the cytoplasm and vacuole when grown under a variety of NaCl concentrations. These results indicate a higher tolerance of Debaryomyces to high internal Na(+), and, in addition, suggest the existence of a transporter driving Na(+) into the vacuole. Moreover, a gene encoding a Na(+) (K(+))/H(+) antiporter from D. hansenii was cloned and sequenced. The gene, designated DhNHX1, exhibited significant homology with genes of the NHE/NHX family. DhNHX1 expression was induced neither at low pH nor by extracellular NaCl. A mutant of S. cerevisiae lacking its own Na(+) transporters (ena1-4Delta nha1 Delta nhx1 Delta), when transformed with DhNHX1, partially recovered cation tolerance as well as the ability to accumulate Na(+) and K(+) into the vacuole. Our analysis provides evidence that DhNhx1p transports Na(+) (and K(+)) into the vacuole and that it can play an important role in ion homeostasis and salt tolerance.