Functional and Structural Role of Amino Acid Residues in the Even-numbered Transmembrane α-Helices of the Bovine Mitochondrial Oxoglutarate Carrier

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Background: Substrate binding and transport mechanisms of mitochondrial carriers are inadequately understood. Results: The effect of mutations on substrate specificity and transport activity was assessed in two human ornithine carrier isoforms. Conclusion:The substrate specificity and transport rate of the two isoforms are defined by a few residues and can be swapped. Significance: The results show how small substrates can trigger transport in carriers.

Section: Discussionmentioning

confidence: 52%

Substrate Specificity of the Two Mitochondrial Ornithine Carriers Can Be Swapped by Single Mutation in Substrate Binding Site

Monné

Miniero

Daddabbo

et al. 2012

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“…The amount of protein incorporated into liposomes was measured as described (22) and was about 25% of the protein added to the reconstitution mixture. The homology model of Oac1p was built based upon the structure of the bovine ADP/ ATP carrier as described (23,24). An anaerobic Oxoid jar was used to grow yeast strains in anaerobiosis.…”

Section: Methodsmentioning

confidence: 99%

α-Isopropylmalate, a Leucine Biosynthesis Intermediate in Yeast, Is Transported by the Mitochondrial Oxalacetate Carrier

Marobbio

Giannuzzi

Paradies

et al. 2008

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In Saccharomyces cerevisiae, ␣-isopropylmalate (␣-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 ␣-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 ␣-IPM transporter. Transport was saturable with a half-saturation constant of 75 ؎ 4 M for ␣-IPM and 0.31 ؎ 0.04 mM for ␤-IPM and was inhibited by the substrates of Oac1p. Though not transported, ␣-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, ␣-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an ␣-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 ␣-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant ⌬OAC1⌬LEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of ⌬OAC1⌬LEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates ␣-ketoisocaproate and ␣-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 ␣-IPM, probably in exchange for oxalacetate.

“…Sequence homology shows that almost every MCF member contains a Lys or an Arg at the position equivalent to MFT Arg-288 (9). This (K/R) residue is required for transport in every MCF protein in which it has been mutated (11,33,34); we also showed MFT Arg-288 to be required for folate transport (Fig. 5B).…”

Section: Discussionmentioning

confidence: 54%

“…Although almost all MCF transporters carry a (D/E) at MFT 142, we found that the MFT absolutely cannot function with a (D/E) residue at this position. In addition to the Trp substitution definitive of this subfamily, all P(I/L)W transporters contain a Gly at MFT 91, whereas the closely related MCF nucleotide transporters and several other MCF proteins require a Lys or an Arg at this position for function (11,33,34). Gly-91 was predicted to be very close to Trp-142 at the base of the cavity.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, other MCF transporters have been shown to require a basic residue for transport function at the position homologous to hamster MFT Gly-91 (11,33,34), and we questioned whether the small size and uncharged nature of this residue were essential for function of the MFT. We also used mutagenesis to query the presence of and functional requirement for a proposed -cation interaction between Trp-142 and Arg-249 as well as to probe position 142 for the requirement of charge, size, and aromatic character.…”

Section: Simulation Of the Descent Of Thf Into The Transport Cavity Omentioning

confidence: 99%

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Tetrahydrofolate Recognition by the Mitochondrial Folate Transporter

Lawrence

Hackett

Moran

2011

A mitochondrial carrier family (MCF) of transport proteins facilitates the transfer of charged small molecules across the inner mitochondrial membrane. The human genome has ϳ50 genes corresponding to members of this family. All MCF proteins contain three repeats of a characteristic and conserved PX(D/E)XX(K/R) motif thought to be central to the mechanism of these transporters. The mammalian mitochondrial folate transporter (MFT) is one of a few MCF members, known as the P(I/L)W subfamily, that have evolved a tryptophan residue in place of the (D/E) in the second conserved motif; the function of this substitution (Trp-142) is unclear. Molecular dynamics simulations of the MFT in its explicit membrane environment identified this tryptophan, as well as several other residues lining the transport cavity, to be involved in a series of sequential interactions that coordinated the movement of the tetrahydrofolate substrate within the transport cavity. We probed the function of these residues by mutagenesis. The mutation of every residue identified by molecular dynamics to interact with tetrahydrofolate during simulated transit into the aqueous channel severely impaired folate transport. Mutation of the subfamily-defining tryptophan residue in the MFT to match the MCF consensus at this position (W142D) was incompatible with cell survival. These studies indicate that MFT Trp-142, as well as other residues lining the transporter interior, coordinate tetrahydrofolate descent and positioning of the substrate in the transporter basin. Overall, we identified residues in the walls and at the base of the transport cavity that are involved in substrate recognition by the MFT.Folate metabolism is essential for mammalian cell survival and consists of reaction sequences that occur in the mitochondrial and cytosolic compartments. In dividing cells, cytosolic folate metabolism uses one-carbon units for purine nucleotide and thymidylate synthesis, whereas mitochondrial folate metabolism primarily drives the supply of formate to the cytoplasm for these processes. Mitochondrial folates are also used as cofactors for mitochondrial glycine synthesis, and cells deficient in mitochondrial folate metabolism are auxotrophic for glycine despite the fact that enzymes capable of glycine synthesis are present in both compartments (1-5). An example of such glycine auxotrophy is the Chinese hamster ovary (CHO)-derived glyB cell, which retains wild-type activity of all the enzymes involved in mitochondrial folate metabolism but is unable to accumulate mitochondrial folates (1-3). Previously, we used complementation of glycine auxotrophy with a retroviral cDNA library to isolate the gene defective in glyB cells; it encoded a protein that transported folates into mitochondria, the mitochondrial folate transporter (MFT) 2 (3). In glyB cells, the MFT open reading frame has a G192E missense mutation that diminishes mitochondrial folate levels by ϳ99% and effectively eliminates the transport of mitochondrial folates (2, 3). Transfection of cDNAs encoding ...