RESUMO
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 genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 15 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates and cofactors across the inner membrane. Here the identification of the mitochondrial carrier for the essential cofactor thiamine pyrophosphate (ThPP) is described. The protein has been overexpressed in bacteria, reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, cells lacking the gene for this carrier had reduced levels of ThPP in their mitochondria, and decreased activity of acetolactate synthase, a ThPP-requiring enzyme found in the organellar matrix. They also required thiamine for growth on fermentative carbon sources.
