A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans

Abstract

Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150-kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, and silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.

Fig. 1

Mpc1 and Mpc2 are evolutionarily conserved mitochondrial inner-membrane proteins. (A) Mpc1 labeled with green fluorescent protein (Mpc1-GFP) and mitochondrial targeted red fluorescent protein (MtRFP) coexpressed in yeast cells. DIC, differential interference contrast. (B) Intact mitochondria, hypotonic-swollen mitoplasts, and TritonX-100–solubilized mitochondria from a strain expressing Mpc1-V5 and Mpc2-His₆/HA₂ with (+) or without (−) proteinase K incubation. An immunoblot of extracts using the indicated antibodies with the whole-cell lysate (WCL) and postmitochondrial supernatant (PMS) is shown. Mge1, Cyb2, and Fzo1 are matrix, intermembrane space, and outer-membrane proteins, respectively. (C) Immunoprecipitations from mitochondrial extracts from mpc1Δ mpc2Δ cells expressing Mpc1 and Mpc2 tagged as indicated. Immunoblot of either immunoprecipitate (IP:HA) or input is shown (HA, hemagglutinin). QCR1 and 2 (ubiquinol–cytochrome c reductase complex core protein 1 and 2) along with Cox II (cytochrome c oxidase subunit 2) are controls for the specificity of the immunoprecipitation. (D) Serial dilutions of the indicated yeast strains spotted on synthetic media lacking leucine and grown at 30°C for 24 hours. (E) Serial dilutions of indicated strains spotted on synthetic media lacking leucine and grown at 30°C for 48 hours. wt, wild type; EV, empty vector.

Fig. 2

dMPC1 is required for pyruvate metabolism in Drosophila. (A) Percentage of living control (dMPC1⁺) or dMPC1 mutant (dMPC⁻) flies after transfer to standard laboratory medium (std. food) or to media containing only sugar. (B) Percentage of living dMPC1⁺ or dMPC⁻ flies carrying the indicated GAL4 and UAS transgenes on sugar media after 8 days. (C to E) Relative concentration of ATP (C), trehalose (D), and glucose (E) in extracts from dMPC1⁺ or dMPC⁻ flies on the indicated diet after either 2 days (D and E) or 3 days (C). (F) Relative abundance of pyruvate and TCA cycle intermediates in dMPC1⁺ or dMPC⁻ flies after 2 days on the indicated diet as measured by gas chromatography–mass spectrometry. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test). Data are shown as mean ± SEM.

Fig. 3

MPC1 is required for mitochondrial pyruvate uptake. (A) Relative abundance of pyruvate in the indicated strains. P values relative to wt. (B) Relative abundance of acetyl-CoA and CoA in the indicated strains. (C) Mitochondrial pyruvate dehydrogenase activity in the indicated strains. P value relative to wt and mpc1Δ. (D) Serial dilutions of the indicated strain on glucose medium grown at 30°C for 48 hours. (E) Uptake of ¹⁴C-pyruvate into mitochondria purified from either wt or mpc1Δ cells containing the indicated plasmid. P value relative to wt + EV and mpc1Δ + MPC1. (F) Mae1Δ mpc1Δ cells transformed with the indicated plasmid and plated on media containing or lacking combinations of leucine or UK-5099. (G) Uptake of ¹⁴C-pyruvate into mitochondria isolated from the mpc1Δ strain containing the indicated plasmid in the presence or absence of UK-5099. ***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant (Student’s t test). Data are shown as mean ± SEM.

Fig. 4

Mammalian MPC1 and MPC2 are required for normal pyruvate metabolism. (A and B) Pyruvate-driven respiration in mouse embryonic fibroblasts under basal and FCCP-stimulated conditions in cells transfected with either control (Cont) small interfering RNAs (siRNAs) or three different siRNAs (si 1–3) targeted to either MPC1 (A) or MPC2 (B). P values relative to control. (C) Pedigrees of families 1, 2, and 3. Circles indicate females; squares, males; and diamonds, unknown sex. Black indicates deceased and white, living. Arrows mark individuals from whom fibroblasts were obtained. (D) The protein region of MPC1 containing the predicted amino acid substitutions from all three families aligned by ClustalW. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. Saccharomyces cerevisiae, Drosophila melanogaster, Homo sapiens, Caenorhabditis elegans, Danio rerio, Arabidopsis thaliana. (E) Pyruvate- (left) and glutamine- (right) supported respiration of fibroblasts harboring either L79H or R97W MPC1 mutations. (F) Pyruvate-supported respiration of either a control or an L79H patient cell line after transduction with the indicated vector. (G) Pyruvate-supported respiration of either a control or an R97W patient cell line after transduction with the indicated vector. (H) Serial dilutions of wt or mae1Δ mpc1Δ yeast strains carrying the indicated plasmid grown on medium lacking uracil for plasmid selection at 30°C for 40 hours. Both long (L) and short (S) forms of R97W were used (with or without exon 4). ***P < 0.001, **P < 0.01, *P < 0.05, †P < 0.10; NS, not significant (Student’s t test). Data are shown as mean ± SEM.