RESUMO
The Ygr125w was previously identified as a vacuolar membrane protein by a proteomic analysis. We found that vacuolar levels of basic amino acids drastically decreased in ygr125wΔ cells. Since N- or C-terminally tagged Ygr125w was not functional, an expression plasmid of YGR125w with HA3-tag inserted in its N-terminal hydrophilic region was constructed. Introduction of this plasmid into ygr125w∆ cells restored the vacuolar levels of basic amino acids. We successfully detected the uptake activity of arginine by the vacuolar membrane vesicles depending on HA3-YGR125w expression. A conserved aspartate residue in the predicted first transmembrane helix (D223) was indispensable for the accumulation of basic amino acids. YGR125w has been recently reported as a gene involved in vacuolar storage of arginine; and it is designated as VSB1. Taken together, our findings indicate that Ygr125w/Vsb1 contributes to the uptake of arginine into vacuoles and vacuolar compartmentalization of basic amino acids.
Assuntos
Aminoácidos Básicos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Arginina/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Transporte Biológico , Clonagem Molecular , Corantes Fluorescentes/química , Expressão Gênica , Teste de Complementação Genética , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana Transportadoras/genética , Plasmídeos/química , Plasmídeos/metabolismo , Compostos de Piridínio/química , Compostos de Amônio Quaternário/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The stm1+ (SPAC17C9.10) gene of Schizosaccharomyces pombe is closely related to genes encoding vacuolar PQ-loop proteins, Ypq1, Ypq2, and Ypq3, of Saccharomyces cerevisiae. When stm1+ fused with GFP was expressed in fission or budding yeast, Stm1-GFP localized at the vacuolar membrane. Isolated vacuolar membrane vesicles from S. cerevisiae cells overexpressing stm1+ exhibited stm1+-dependent arginine and lysine uptake activity. Exchange activity of arginine and histidine/arginine, as observed for Ypq2 of S. cerevisiae, was also detected in the vesicles expressing stm1+. The expression levels of stm1+ in S. pombe cells significantly affected the vacuolar contents of lysine, histidine, and arginine. These results suggest that Stm1 is a vacuolar PQ-loop protein involved in the transport of basic amino acids across the vacuolar membrane.
Assuntos
Arginina/metabolismo , Membranas Intracelulares/metabolismo , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Vacúolos/metabolismo , Arginina/genética , Transporte Biológico Ativo/fisiologia , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lisina/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Vacúolos/genéticaRESUMO
In nutrient-rich conditions, basic amino acids are actively accumulated into the vacuoles by H+-coupled transporters in Saccharomyces cerevisiae. In addition to the H+-coupled systems, the existence of an exchanger for arginine and histidine was indicated by kinetic analysis using isolated vacuolar membrane vesicles; however, the gene(s) involved in the activity has not been identified. Here, we show that the uptake activity of arginine driven by an artificially imposed histidine gradient decreased significantly by the disruption of the gene encoding vacuolar PQ-loop protein Ypq2, but not by those of Ypq1 and Ypq3. The exchange activity was restored by the expression of YPQ2. Furthermore, the substitution of a conserved proline residue, Pro29, in Ypq2 greatly decreased the exchange activity. These results suggest that Ypq2 is responsible for the exchange activity of arginine and histidine across the vacuolar membrane, and the conserved proline residue in the PQ-loop motif is required for the activity.