Identification and reconstitution of theyeast mitochondrial transporter for thiamine pyrophosphate

Marobbio, C; Vozza, Angelo; Harding, M.; Bisaccia, Faustino; Palmieri, Ferdinando; Walker, John E.

doi:10.1093/emboj/cdf583

Cited by 91 publications

(107 citation statements)

References 43 publications

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“…Thus, net import of the substrate would occur, as predicted and shown experimentally for the transporters of thiamine pyrophosphate (Fig. S28 in SI Appendix) (39), oxaloacetate ( Fig. S11 in SI Appendix) (40), citrate (Fig.…”

Section: Discussionsupporting

confidence: 58%

The mechanism of transport by mitochondrial carriers based on analysis of symmetry

Robinson

Overy²,

Kunji³

2008

Proc. Natl. Acad. Sci. U.S.A.

200

363

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The structures of mitochondrial transporters and uncoupling proteins are 3-fold pseudosymmetrical, but their substrates and coupling ions are not. Thus, deviations from symmetry are to be expected in the substrate and ion-binding sites in the central aqueous cavity. By analyzing the 3-fold pseudosymmetrical repeats from which their sequences are made, conserved asymmetric residues were found to cluster in a region of the central cavity identified previously as the common substrate-binding site. Conserved symmetrical residues required for the transport mechanism were found at the water-membrane interfaces, and they include the three PX T he members of the mitochondrial transporter or carrier family translocate nucleotides, amino acids, inorganic ions, keto acids, and vitamins across the mitochondrial inner membrane (1, 2). Uncoupling proteins, which generate heat by dissipating the proton electrochemical gradient, also belong to the family (3-5). The amino acid sequences have 3 homologous repeats (6) and a structure with pseudosymmetry (7). Each repeat is folded into 2 transmembrane ␣-helices linked by a short ␣-helix on the matrix side (8) and contains the signature motif PX[DE]XX[RK] (9). The proline residues kink the oddnumbered transmembrane ␣-helices H1, H3, and H5, and the charged residues form a salt-bridge network connecting the C-terminal end of the transmembrane ␣-helices, closing the transporter on the matrix side (8). During the transport cycle, the carriers form the cytoplasmic and matrix states in which the substrate-binding site of the carrier is open to the mitochondrial intermembrane space and matrix, respectively (10). According to the single binding center-gating pore mechanism, interconversion of the 2 conformational states via a transition intermediate leads to substrate translocation (11). In agreement, in the cytoplasmic state, a central substrate-binding site has been identified by applying chemical and distance constraints to comparative models (12, 13). The substrates bind to 3 major sites on the even-numbered ␣-helices, which are related by symmetry and located approximately in the middle of the membrane. In molecular dynamics simulations, ADP binds to the ADP/ATP carrier at the common substrate-binding site (14). The structural changes required for the translocation of the substrate are unknown, but docking of the substrate could disrupt the matrix salt bridge network, allowing the transporter to convert to the matrix state (12, 13). The yeast ADP/ATP carriers function as monomers (15), and other mitochondrial transporters are likely to operate in the same way.Residues that are important for the transport mechanism are likely to be symmetrical, whereas residues involved in substrate binding will be asymmetrical reflecting the asymmetry of the substrates. By scoring the symmetry of residues in the sequence repeats, we have identified the substrate-binding sites and salt bridge networks that are important for the transport mechanism in family members. No preexisting molecular structure i...

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Section: Discussionsupporting

confidence: 58%

The mechanism of transport by mitochondrial carriers based on analysis of symmetry

Robinson

Overy²,

Kunji³

2008

Proc. Natl. Acad. Sci. U.S.A.

200

363

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“…The recent discovery of the Tpc1p gene (5) in yeast (Swiss-Prot entry P53257, YGR096W) suggested another function for SLC25A19. Tpc1p transports ThPP produced in the cytosol into the mitochondria in exchange for mitochondrially generated ThMP, which then is metabolized and converted back to ThPP (5). In an in vitro exchange assay, we found that a protein containing the G177A mutation reduced ThPP and ThMP transport by Ϸ70% compared with wild-type protein.…”

Section: Discussionmentioning

confidence: 87%

“…While this study was underway, a novel mitochondrial transporter, Tpc1p, was discovered in Saccharomyces cerevisiae (5). This protein transports ThPP into the mitochondria in exchange for thiamine monophosphate (ThMP) and is the yeast protein most similar to human SLC25A19.…”

Section: Resultsmentioning

confidence: 99%

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Knockout of Slc25a19 causes mitochondrial thiamine pyrophosphate depletion, embryonic lethality, CNS malformations, and anemia

Lindhurst

Fiermonte

Song

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

145

138

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SLC25A19 mutations cause Amish lethal microcephaly (MCPHA), which markedly retards brain development and leads to ␣-ketoglutaric aciduria. Previous data suggested that SLC25A19, also called DNC, is a mitochondrial deoxyribonucleotide transporter. We generated a knockout mouse model of Slc25a19. These animals had 100% prenatal lethality by embryonic day 12. Affected embryos at embryonic day 10.5 have a neural-tube closure defect with ruffling of the neural fold ridges, a yolk sac erythropoietic failure, and elevated ␣-ketoglutarate in the amniotic fluid. We found that these animals have normal mitochondrial ribo-and deoxyribonucleoside triphosphate levels, suggesting that transport of these molecules is not the primary role of SLC25A19. We identified thiamine pyrophosphate (ThPP) transport as a candidate function of SLC25A19 through homology searching and confirmed it by using transport assays of the recombinant reconstituted protein.The mitochondria of Slc25a19 ؊/؊ and MCPHA cells have undetectable and markedly reduced ThPP content, respectively. The reduction of ThPP levels causes dysfunction of the ␣-ketoglutarate dehydrogenase complex, which explains the high levels of this organic acid in MCPHA and suggests that mitochondrial ThPP transport is important for CNS development.development ͉ mitochondrial transporter ͉ mouse model ͉ thiamine pyrophosphate deficiency ͉ neural tube defect

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“…PHO3 represents a periplasmic acid phosphatase with a high affinity to thiamin phosphates and with an additional capability to tightly bind free thiamin [22]. A TPC1 gene encodes a mitochondrial membrane protein that transports TDP, synthesized de novo in the cytoplasm, into the mitochondria, where it can be incorporated into several essential TDP-dependent enzymes [23]. Our understanding of the genetic regulation of thiamin biosynthesis in yeast is at a very preliminary stage (for a recent review see: [3]), even though it has been known for four decades that the addition of thiamin to the medium represses thiamin synthesis and cellular uptake.…”

Section: The Genetic Control Of Thiamin Biosynthesis In Yeastsmentioning

confidence: 99%

The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts

Kowalska

Kozik

2008

Cellular and Molecular Biology Letters

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Thiamin (vitamin B1) is an essential molecule for all living organisms. Its major biologically active derivative is thiamin diphosphate, which serves as a cofactor for several enzymes involved in carbohydrate and amino acid metabolism. Important new functions for thiamin and its phosphate esters have recently been suggested, e.g. in gene expression regulation by influencing mRNA structure, in DNA repair after UV illumination, and in the protection of some organelles against reactive oxygen species. Unlike higher animals, which rely on nutritional thiamin intake, yeasts can synthesize thiamin de novo. The biosynthesis pathways include the separate synthesis of two precursors, 4-amino -5-hydroxymethyl-2-methylpyrimidine diphosphate and 5-(2-hydroxyethyl)-4-methylthiazole phosphate, which are then condensed into thiamin monophosphate. Additionally, yeasts evolved salvage mechanisms to utilize thiamin and its dephosphorylated late precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole, from the environment. The current state of knowledge on the discrete steps of thiamin biosynthesis in yeasts is far from satisfactory; many intermediates are postulated only by analogy to the much better understood biosynthesis process in bacteria. On the other hand, the genetic mechanisms regulating thiamin biosynthesis in yeasts are currently under extensive exploration. Only recently, the structures of some of the yeast enzymes involved in thiamin biosynthesis, such as thiamin diphosphokinase and thiazole synthase, were determined at the atomic resolution, and mechanistic proposals for the catalysis of particular biosynthetic steps started to emerge.

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Identification and reconstitution of theyeast mitochondrial transporter for thiamine pyrophosphate

Cited by 91 publications

References 43 publications

The mechanism of transport by mitochondrial carriers based on analysis of symmetry

The mechanism of transport by mitochondrial carriers based on analysis of symmetry

Knockout of Slc25a19 causes mitochondrial thiamine pyrophosphate depletion, embryonic lethality, CNS malformations, and anemia

The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts

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