RESUMO
The nuclear genes of Saccharomyces cerevisiae YHM2, ODC1 and ODC2 encode three transporters that are localized in the inner mitochondrial membrane. In this study, the roles of YHM2, ODC1 and ODC2 in the assimilation of nitrogen and in the biosynthesis of lysine have been investigated. Both the odc1Δodc2Δ double knockout and the yhm2Δ mutant grew similarly as the YPH499 wild-type strain on synthetic minimal medium (SM) containing 2% glucose and ammonia as the main nitrogen source. In contrast, the yhm2Δodc1Δodc2Δ triple knockout exhibited a marked growth defect under the same conditions. This defect was fully restored by the individual expression of YHM2, ODC1 or ODC2 in the triple deletion strain. Furthermore, the lack of growth of yhm2Δodc1Δodc2Δ on 2% glucose SM was rescued by the addition of glutamate, but not glutamine, to the medium. Using lysine-prototroph YPH499-derived strains, the yhm2Δodc1Δodc2Δ knockout (but not the odc1Δodc2Δ and yhm2Δ mutants) also displayed a growth defect in lysine biosynthesis on 2% glucose SM, which was rescued by the addition of lysine and, to a lesser extent, by the addition of 2-aminoadipate. Additional analysis of the triple mutant showed that it is not respiratory-deficient and does not display mitochondrial DNA instability. These results provide evidence that only the simultaneous absence of YHM2, ODC1 and ODC2 impairs the export from the mitochondrial matrix of i) 2-oxoglutarate which is necessary for the synthesis of glutamate and ammonium fixation in the cytosol and ii) 2-oxoadipate which is required for lysine biosynthesis in the cytosol. Finally, the data presented allow one to suggest that the yhm2Δodc1Δodc2Δ triple knockout is suitable in complementation studies aimed at assessing the pathogenic potential of human SLC25A21 (ODC) mutations.
Assuntos
Compostos de Amônio/metabolismo , Meios de Cultura/síntese química , Lisina/biossíntese , Proteínas de Transporte da Membrana Mitocondrial/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Meios de Cultura/química , Transportadores de Ácidos Dicarboxílicos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Técnicas de Inativação de Genes , Glutamatos/farmacologia , Glutamina/farmacologia , Lisina/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The genome of Saccharomyces cerevisiae encodes 35 putative members of the mitochondrial carrier family. Known members of this family transport substrates and products across the inner membranes of mitochondria. We are attempting to identify the functions of the yeast mitochondrial transporters via high-yield expression in Escherichia coli and/or S. cerevisiae, purification and reconstitution of their protein products into liposomes, where their transport properties are investigated. With this strategy, we have already identified the functions of seven S. cerevisiae gene products, whose structural and functional properties assigned them to the mitochondrial carrier family. The functional information obtained in the reconstituted system and the use of knock-out yeast strains can be usefully exploited for the investigation of the physiological role of individual transporters. Furthermore, the yeast carrier sequences can be used to identify the orthologous proteins in other organisms, including man.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana Transportadoras , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos Básicos , Animais , Antiporters/química , Antiporters/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Carnitina Aciltransferases/química , Carnitina Aciltransferases/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Clonagem Molecular , Transportadores de Ácidos Dicarboxílicos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Genes , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Química Encefálica , Púrpura de Bromocresol/farmacologia , Células CHO , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Ligação ao Cálcio/fisiologia , Clonagem Molecular , Cricetinae , Citosol/metabolismo , DNA Complementar/genética , Escherichia coli , Etiquetas de Sequências Expressas , Humanos , Taninos Hidrolisáveis/farmacologia , Moduladores de Transporte de Membrana , Proteínas de Membrana Transportadoras/antagonistas & inibidores , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Membrana Transportadoras/fisiologia , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/isolamento & purificação , Proteínas Mitocondriais/fisiologia , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Especificidade de Órgãos , Filogenia , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier protein family, most of which have not yet been functionally identified. Here the identification of the mitochondrial carrier for S-adenosylmethionine (SAM) Sam5p is described. The corresponding gene has been overexpressed in bacteria and the protein has been reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, (i) the Sam5p-GFP protein was found to be targeted to mitochondria; (ii) the cells lacking the gene for this carrier showed auxotrophy for biotin (which is synthesized in the mitochondria by the SAM-requiring Bio2p) on fermentable carbon sources and a petite phenotype on non-fermentable substrates; and (iii) both phenotypes of the knock-out mutant were overcome by expressing the cytosolic SAM synthetase (Sam1p) inside the mitochondria.
Assuntos
Mitocôndrias/metabolismo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/metabolismo , Biotina/metabolismo , Genes Reporter , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Mitocondriais , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The nuclear genome of Saccharomyces cerevisiae encodes 35 members of a family of membrane proteins. Known members transport substrates and products across the inner membranes of mitochondria. We have localized two hitherto unidentified family members, Odc1p and Odc2p, to the inner membranes of mitochondria. They are isoforms with 61% sequence identity, and we have shown in reconstituted liposomes that they transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a strict counter exchange mechanism. Intraliposomal adipate and glutarate and to a lesser extent malate and citrate supported [14C]oxoglutarate uptake. The expression of Odc1p, the more abundant isoform, made in the presence of nonfermentable carbon sources, is repressed by glucose. The main physiological roles of Odc1p and Odc2p are probably to supply 2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix to the cytosol where they are used in the biosynthesis of lysine and glutamate, respectively, and in lysine catabolism.
Assuntos
Adipatos/metabolismo , Proteínas de Transporte/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mitocôndrias/metabolismo , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Recombinantes/metabolismo , Frações Subcelulares/metabolismoRESUMO
Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein. The transport specificities of some family members are known, but most are not. The function of the OAC has been revealed by overproduction in Escherichia coli, reconstitution into liposomes, and demonstration that the proteoliposomes transport malonate, oxaloacetate, sulfate, and thiosulfate. Reconstituted OAC catalyzes both unidirectional transport and exchange of substrates. In S. cerevisiae, OAC is in inner mitochondrial membranes, and deletion of its gene greatly reduces transport of oxaloacetate sulfate, thiosulfate, and malonate. Mitochondria from wild-type cells swelled in isoosmotic solutions of ammonium salts of oxaloacetate, sulfate, thiosulfate, and malonate, indicating that these anions are cotransported with protons. Overexpression of OAC in the deletion strain increased greatly the [(35)S]sulfate/sulfate and [(35)S]sulfate/oxaloacetate exchanges in proteoliposomes reconstituted with digitonin extracts of mitochondria. The main physiological role of OAC appears to be to use the proton-motive force to take up into mitochondria oxaloacetate produced from pyruvate by cytoplasmic pyruvate carboxylase.