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IDENTIFICATION AND FUNCTIONAL RECONSTITUTION OF THE YEAST MITOCHONDRIAL CARRIER FOR S-ADENOSYLMETHIONINE
No full textIn Saccharomyces cerevisiae, S-adenosylmethionine (SAM) is synthesized from ATP and methionine by two synthetases, Samlp and Sam2p, that are both localized exclusively in the cytosol. SAM must therefore be imported into the mitochondria, where it is required as a methyl group donor for DNA, RNA, protein and sterol methylation and as an essential cofactor in the last steps of biotin and lipoic acid biosynthesis catalyzed by biotin synthetase (Bio2p) and lipoate synthetase (Lip5p), respectively. Here we report the identification and functional characterization of the mitochondrial SAM carrier (Sam5p) encoded by YNL003c, also known as PET8. Sam5p is 284 amino acids long and has the characteristic sequence features of the mitochondrial carrier family. Sam5p was overexpressed in bacteria, purified and reconstituted into phospholipid vesicles. It transports SAM, S¬adenosylhomocysteine, and to a lesser extent, the nonphysiological structurally¬-related compounds S-adenosylcysteine and sinefungin, but none of the many other compounds tested. SAM is transported by Sam5p either by uniport or by exchange with S-adenosylhomocysteine, which is produced from SAM in methylation reactions inside mitochondria. The green fluorescent protein (GFP) fused to Sam5p was found to be targeted to mitochondria. Cells lacking the gene for this carrier (sam5Δ cells) 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. Furthermore, both phenotypes of the sam5Δ cells were restored by directing the cytosolic SAM synthetase (Sam1p) into the mitochondria
Identification of the yeast ACR1 gene product as a succinate-fumarate transporter essential for growth on ethanol or acetate
No full textIdentification of the yeast ACR1 gene product as a succinate-fumarate transporter essential for growth on ethanol or acetate
No full textThe protein encoded by the ACR1 gene in Saccharomyces cerevisiae belongs to a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 28 members of the family are unknown. The yeast ACR1 gene was introduced into Escherichia coli on an expression plasmid. The protein was over-produced as inclusion bodies, which were purified and solubilised in the presence of sarkosyl. The solubilised protein was reconstituted into liposomes and shown to transport fumarate and succinate. Its physiological role in S. cerevisiae is probably to transport cytoplasmic succinate, derived from isocitrate by the action of isocitrate lyase in the cytosol, into the mitochondrial matrix in exchange for fumarate. This exchange activity and the subsequent conversion of fumarate to oxaloacetate in the cytosol would be essential for the growth of S. cerevisiae on ethanol or acetate as the sole carbon source
Mitochondrial glutamate carrier GC1 as a newly identified player in the control of glucose-stimulated insulin secretion
No full textThe SLC25 carrier family mediates solute transport across the inner mitochondrial membrane, a process that is still poorly characterized regarding both the mechanisms and proteins implicated. This study investigated mitochondrial glutamate carrier GC1 in insulin-secreting β-cells. GC1 was cloned from insulin-secreting cells, and sequence analysis revealed hydropathy profile of a six-transmembrane protein, characteristic of mitochondrial solute carriers. GC1 was found to be expressed at the mRNA and protein levels in INS-1E β-cells and pancreatic rat islets. Immunohistochemistry showed that GC1 was present in mitochondria, and ultrastructural analysis by electron microscopy revealed inner mitochondrial membrane localization of the transporter. Silencing of GC1 in INS-1E β-cells, mediated by adenoviral delivery of short hairpin RNA, reduced mitochondrial glutamate transport by 48% (p < 0.001). Insulin secretion at basal 2.5mM glucose and stimulated either by intermediate 7.5 mM glucose or non-nutrient 30 mM KCl was not modified by GC1 silencing. Conversely, insulin secretion stimulated with optimal 15 mM glucose was reduced by 23% (p < 0.005) in GC1 knocked down cells compared with controls. Adjunct of cell-permeant glutamate (5mM dimethyl glutamate) fully restored the secretory response at 15 mM glucose (p < 0.005). Kinetics of insulin secretion were investigated in perifused isolated rat islets. GC1 silencing in islets inhibited the secretory response induced by 16.7 mM glucose, both during first (-25%, p<0.05) and second (-33%, p<0.05) phases. This study demonstrates that insulin-secreting cells depend on GC1 for maximal glucose response, thereby assigning a physiological function to this newly identified mitochondrial glutamate carrier. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc
Identification and characterization of the mitochondrial transporter for S-adenosylmethionine in yeast and man
No full textThe mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttles metabolites and cofactors through this membrane, and connect cytoplasm functions with others in the matrix. In S. cerevisiae, S-adenosylmethionine (SAM) is synthesized from ATP and methionine by two synthesases, Sam1p and Sam2p, that are both localized exclusively in the cytosol (1). SAM has therefore to be imported into the mitochondria, where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins, and is required as essential cofactor in the last step of biotin and lipoic acid biosyntheses, which are catalyzed by biotin synthetase (Bio2p) and lipoate synthetatse (Lip5p), respectively. Here we report the identification and functional characterization of the yeast mitochondrial SAM carrier (Sam5p), encoded by YNL003c also known as PET8(2), and of its human ortholog named SAMC, encoded by SLC25A26
S. Cerevisiae ACR1 gene encodes a novel mitochondrial carrier essential for growth on ethanol or acetate
No full textMitochondrial Carriers for Aspartate, Glutamate and Other Amino Acids: A Review
Full text linkMembers of the mitochondrial carrier (MC) protein family transport various molecules across the mitochondrial inner membrane to interlink steps of metabolic pathways and biochemical processes that take place in different compartments; i.e., are localized partly inside and outside the mitochondrial matrix. MC substrates consist of metabolites, inorganic anions (such as phosphate and sulfate), nucleotides, cofactors and amino acids. These compounds have been identified by in vitro transport assays based on the uptake of radioactively labeled substrates into liposomes reconstituted with recombinant purified MCs. By using this approach, 18 human, plant and yeast MCs for amino acids have been characterized and shown to transport aspartate, glutamate, ornithine, arginine, lysine, histidine, citrulline and glycine with varying substrate specificities, kinetics, influences of the pH gradient, and capacities for the antiport and uniport mode of transport. Aside from providing amino acids for mitochondrial translation, the transport reactions catalyzed by these MCs are crucial in energy, nitrogen, nucleotide and amino acid metabolism. In this review we dissect the transport properties, phylogeny, regulation and expression levels in different tissues of MCs for amino acids, and summarize the main structural aspects known until now about MCs. The effects of their disease-causing mutations and manipulation of their expression levels in cells are also considered as clues for understanding their physiological functions
Identification of the mitochondrial ATP-Mg/Pi transporter: bacterial expression, reconstitution, functional characterization and tissue distribution
No full textThe mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites, nucleotides, and cofactors through this membrane and thereby connect and/or regulate cytoplasm and matrix functions. ATP-Mg is transported in exchange for phosphate, but no protein has ever been associated with this activity. We have isolated three human cDNAs that encode proteins of 458, 468, and 489 amino acids with 66-75% similarity and with the characteristic features of the mitochondrial carrier family in their C-terminal domains and three EF-hand Ca(2+)-binding motifs in their N-terminal domains. These proteins have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties and their targeting to mitochondria demonstrate that they are isoforms of the ATP-Mg/Pi carrier described in the past in whole mitochondria. The tissue specificity of the three isoforms shows that at least one isoform was present in all of the tissues investigated. Because phosphate recycles via the phosphate carrier in mitochondria, the three isoforms of the ATP-Mg/Pi carrier are most likely responsible for the net uptake or efflux of adenine nucleotides into or from the mitochondria and hence for the variation in the matrix adenine nucleotide content, which has been found to change in many physiopathological situations
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