RESUMO
Using PCR, reverse transcription-PCR (RT-PCR) and colony hybridization in a genomic library, we isolated six genes which encode type II P-type ATPases in Neurospora crassa. The six full-length cDNAs were cloned in a yeast expression vector and transformed into Saccharomyces cerevisiae null Ca2+- or Na+-ATPase mutants. Three cDNAs suppressed the defect of the Ca2+ mutant and two of these protected from Mn2+ toxicity. One cDNA suppressed the defect of the Na+ mutant and two cDNAs were not functional in S. cerevisiae. The expression of the transcripts of the six genes in the presence of Ca2+, Na+, high pH or supporting an osmotic shock indicated that, with the exception of one of the Ca2+-ATPases, the main function of the cloned ATPases is the adaptation to stress conditions. The relationship between the cloned fungal Ca2+- and Na+-ATPases and plant type II P-ATPases is discussed.
Assuntos
Adenosina Trifosfatases/genética , ATPases Transportadoras de Cálcio/genética , Proteínas de Transporte de Cátions , Neurospora crassa/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/metabolismo , Clonagem Molecular , Primers do DNA , DNA Complementar , Dados de Sequência Molecular , Neurospora crassa/metabolismo , Biossíntese de Proteínas , Homologia de Sequência de AminoácidosRESUMO
A cDNA library in a yeast expression vector was prepared from roots of Arabidopsis exposed to salt and was used to select Li(+)-tolerant yeast transformants. The cDNA SAL1 isolated from one of these transformants encodes a polypeptide of 353 amino acid residues. This protein is homologous to the HAL2 and CysQ phosphatases of yeast and Escherichia coli, respectively. Partial cDNA sequences in the data bases indicate that rice produces a phosphatase highly homologous to SAL1 and that a second gene homologous to SAL1 exists in Arabidopsis. The SAL1 protein expressed in E. coli showed 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities. In yeast, SAL1 restored the ability of a hal2/met22 mutant to grow on sulfate as a sole sulfur source, increased the intracellular Li+ tolerance, and modified Na+ and Li+ effluxes. We propose that the product of SAL1 participates in the sulfur assimilation pathway as well as in the phosphoinositide signaling pathway and that changes in the latter may affect Na+ and Li+ fluxes.
Assuntos
Proteínas de Arabidopsis , Genes de Plantas , Nucleotidases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Zea mays/enzimologia , Zea mays/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar , Biblioteca Gênica , Cinética , Lítio/farmacologia , Magnésio/farmacologia , Dados de Sequência Molecular , Nucleotidases/biossíntese , Nucleotidases/genética , Oryza/enzimologia , Monoéster Fosfórico Hidrolases/biossíntese , Monoéster Fosfórico Hidrolases/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Sódio/farmacologiaRESUMO
The ENA2 gene encoding a P-type ATPase involved in Na+ and Li+ effluxes in Saccharomyces cerevisiae has been isolated. The putative protein encoded by ENA2 differs only in thirteen amino acids from the protein encoded by ENA1/PMR2. However, ENA2 has a very low level of expression and for this reason did not confer significant Li+ tolerance on a Li+ sensitive strain. ENA1 and ENA2 are the first two units of a tandem array of four highly homologous genes with probably homologous functions.
Assuntos
Adenosina Trifosfatases/genética , Regulação Enzimológica da Expressão Gênica , Genes Fúngicos/genética , Isoenzimas/genética , Saccharomyces cerevisiae/genética , Sódio/metabolismo , Sequência de Bases , DNA Recombinante , Resistência Microbiana a Medicamentos , Óperon Lac/genética , Lítio/metabolismo , Lítio/farmacologia , Dados de Sequência Molecular , Plasmídeos/genéticaRESUMO
The gene ENA1 was cloned by its ability to complement the Li+ sensitivity of a low Li(+)-efflux strain. The nucleotide sequence of the cloned DNA fragment showed that there are two almost identical genes in tandem, and predicts that they encode P-ATPases. Disruption of both genes originated a strain defective in Na+ and Li+ effluxes, and sensitive to Na+, to Li+ and to alkaline pH. By transformation with ENA1 the defective effluxes and tolerances were repaired.
