RESUMO
The mitochondrial phosphate transporter of Saccharomyces cerevisiae, encoded by MIR1 (YJR077C) gene, shows divergence among the transporters in various eukaryotes. We have characterized another gene, YER053C, that appeared to encode an orthologous mitochondrial phosphate transporter of yeast. The predicted amino acid sequence of the YER053C protein is much more similar to that of mitochondrial phosphate transporters of other species than that of MIR1. RNA gel blot analysis indicated that, like the MIR1 promoter, the YER053C promoter is functional and that its activity varies according to aeration. An MIR1 gene null mutant did not grow on glycerol medium, whereas a YER053C null mutant grew well on the medium, suggesting that the YER053C gene is not essential for the mitochondrial function. YER053C also did not support the growth of the MIR1 null mutant on glycerol. The MIR1 and YER053C proteins were expressed in Escherichia coli and then reconstituted into liposomes. Unlike the proteoliposomes of MIR1, those of YER053C did not exhibit significant phosphate transport activity. Unexpectedly, it was shown that YER053C is localized in vacuoles, not mitochondria, by immunological electron microscopy. These results suggest that, during evolution, yeast lost the function and/or mitochondrial targeting of YER053C and then recruited an atypical MIR1 as the only transporter.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Expressão Gênica/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Proteínas de Transporte de Fosfato , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Vacúolos/metabolismo , Sequência de Aminoácidos/genética , Animais , Divisão Celular/genética , Divisão Celular/fisiologia , Escherichia coli/genética , Evolução Molecular , Humanos , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Mutação/genética , Mutação/fisiologia , Fosforilação Oxidativa , Proteínas de Ligação a Fosfato , Filogenia , Plantas/genética , Regiões Promotoras Genéticas/genética , Proteolipídeos/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , Vacúolos/ultraestruturaRESUMO
cDNA clones encoding mitochondrial phosphate transporters were isolated from four herbaceous plants. The cDNAs for the soybean, maize and rice transporters contained entire coding regions, whereas the Arabidopsis cDNA lacked the 5' portion. The hydropathy profiles of the deduced amino acid sequences predicted the existence of six membrane-spanning domains which are highly conserved in the mitochondrial transporter family. In soybeans, the mRNA level for the transporter was high in tissues containing dividing cells. It was suggested that there are multiple copies of transporter genes in both dicots and monocots. The soybean transporter was expressed as inclusion bodies in Escherichia coli, solubilized with detergents, and then reconstituted into liposomes. The resulting proteoliposomes exhibited high phosphate transport activity. The activity was inhibited by N-ethylmaleimide, like those of mammalian phosphate transporters.