Identification by bacterial expression and functional reconstitution of the yeast genomic sequence encoding the mitochondrial dicarboxylate carrier protein
ABSTRACT The inner membranes of mitochondria contain a family of transport proteins of related sequence and structure. The DNA sequence of the genome of Saccharomyces cerevisiae encodes at least 35 members of this family. Three of them can be recognised as known isoforms of the ADP-ATP translocase and two others as the phosphate and citrate carriers. The transport functions of the remainder cannot be identified with certainty. One of them, encoded on yeast chromosome xii, shows a fairly close sequence relationship to the known sequence of the bovine mitochondrial oxoglutarate-malate carrier. The yeast protein has been obtained by over-expression in Escherichia coli, reconstituted into phospholipid vesicles and shown to have transport properties characteristic of the mitochondrial carrier for dicarboxylate ions, such as malate, and also phosphate, previously biochemically characterised, but not sequenced, from both mammalian and yeast mitochondria. This is the first example of the biochemical identification of an unknown membrane protein encoded in the yeast genome since the completion of the genomic sequence.
Article: alpha-Isopropylmalate, a leucine biosynthesis intermediate in yeast, is transported by the mitochondrial oxalacetate carrier.[show abstract] [hide abstract]
ABSTRACT: In Saccharomyces cerevisiae, alpha-isopropylmalate (alpha-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported alpha-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di- and tricarboxylate transporters as well as the previously proposed alpha-IPM transporter. Transport was saturable with a half-saturation constant of 75 +/- 4 microm for alpha-IPM and 0.31 +/- 0.04 mm for beta-IPM and was inhibited by the substrates of Oac1p. Though not transported, alpha-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, alpha-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an alpha-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with alpha-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant DeltaOAC1DeltaLEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of DeltaOAC1DeltaLEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates alpha-ketoisocaproate and alpha-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of alpha-IPM, probably in exchange for oxalacetate.Journal of Biological Chemistry 09/2008; 283(42):28445-53. · 4.77 Impact Factor
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ABSTRACT: Mitochondria play a key role in respiration and energy production and are involved in multiple eukaryotic but also in several plant specific metabolic pathways. Solute carriers in the inner mitochondrial membrane connect the internal metabolism with that of the surrounding cell. Because of their common basic structure, these transport proteins affiliate to the mitochondrial carrier family (MCF). Generally, MCF proteins consist of six membrane spanning helices, exhibit typical conserved domains and appear as homodimers in the native membrane. Although structurally related, MCF proteins catalyze the specific transport of various substrates, such as nucleotides, amino acids, dicarboxylates, cofactors, phosphate or H(+). Recent investigations identified MCF proteins also in several other cellular compartments and therefore their localization and physiological function is not only restricted to mitochondria. MCF proteins are a characteristic feature of eukaryotes and bacterial genomes lack corresponding sequences. Therefore, the evolutionary origin of MCF proteins is most likely associated with the establishment of mitochondria. It is not clear whether the host cell, the symbiont, or the chimerical organism invented the ancient MCF sequence. Here, we try to explain the establishment of different MCF proteins and focus on the characteristics of members from plants, in particular from Arabidopsis thaliana.Frontiers in plant science. 01/2012; 3:2.
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ABSTRACT: The engulfment of a photoautotrophic cyanobacterium by a primitive mitochondria-bearing eukaryote traces back to more than 1.2 billion years ago. This single endosymbiotic event not only provided the early petroalgae with the metabolic capacity to perform oxygenic photosynthesis, but also introduced a plethora of other metabolic routes ranging from fatty acids and amino acids biosynthesis, nitrogen and sulfur assimilation to secondary compounds synthesis. This implicated the integration and coordination of the newly acquired metabolic entity with the host metabolism. The interface between the host cytosol and the plastidic stroma became of crucial importance in sorting precursors and products between the plastid and other cellular compartments. The plastid envelope membranes fulfill different tasks: they perform important metabolic functions, as they are involved in the synthesis of carotenoids, chlorophylls, and galactolipids. In addition, since most genes of cyanobacterial origin have been transferred to the nucleus, plastidial proteins encoded by nuclear genes are post-translationally transported across the envelopes through the TIC-TOC import machinery. Most importantly, chloroplasts supply the photoautotrophic cell with photosynthates in form of reduced carbon. The innermost bilayer of the plastidic envelope represents the permeability barrier for the metabolites involved in the carbon cycle and is literally stuffed with transporter proteins facilitating their transfer. The intracellular metabolite transporters consist of polytopic proteins containing membrane spans usually in the number of four or more α-helices. Phylogenetic analyses revealed that connecting the plastid with the host metabolism was mainly a process driven by the host cell. In Arabidopsis, 58% of the metabolite transporters are of host origin, whereas only 12% are attributable to the cyanobacterial endosymbiont. This review focuses on the metabolite transporters of the inner envelope membrane of plastids, in particular the electrochemical potential-driven class of transporters. Recent advances in elucidating the plastidial complement of metabolite transporters are provided, with an update on phylogenetic relationship of selected proteins.Frontiers in plant science. 01/2011; 2:50.