A Mitochondrial Pyruvate Carrier Required for Pyruv

NIH Public AccessAuthor ManuscriptScience. Author manuscript; available in PMC 2013 June hed in final edited form as:Science. 2012 July 6; 337(6090): 96–100. doi:10.1126/-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptA Mitochondrial Pyruvate Carrier Required for Pyruvate Uptakein Yeast, Drosophila, and HumansDaniel K. Bricker1,*, Eric B. Taylor2,*, John C. Schell2,*, Thomas Orsak2,*, Audrey Boutron3,Yu-Chan Chen2, James E. Cox4, Caleb M. Cardon2, Jonathan G. Van Vranken2, NoahDephoure5, Claire Redin6, Sihem Boudina7, Steven P. Gygi5, Michèle Brivet3, Carl l1, and Jared Rutter2,†1Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT84112, USA2Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112,USA3Laboratoire de Biochimie, AP-HP Hôpital de Bicêtre, Le Kremlin Bicêtre, France4Metabolomics Core Research Facility, University of Utah School of Medicine, Salt Lake City, UT84112, USA5Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA6Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), Strasbourg, France7Department of Medicine, University of Utah School of Medicine, Salt Lake City, UT 84112, USAAbstractPyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified twoproteins, 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 innermitochondrial membrane. Yeast and

Drosophila mutants lacking

MPC1 display impaired pyruvatemetabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acidcycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, andsilencing of

MPC1 or

MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation inMPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Humangenetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemiarevealed a causal locus that mapped to

MPC1, changing single amino acids that are conservedthroughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of themitochondrial pyruvate te occupies a pivotal node in the regulation of carbon metabolism as it is the endproduct of glycolysis and a major substrate for the tricarboxylic acid (TCA) cycle inmitochondria. Pyruvate lies at the intersection of these catabolic pathways with anabolicpathways for lipid synthesis, amino acid biosynthesis, and gluconeogenesis. As a result, thefailure to correctly partition carbon between these fates lies at the heart of the alteredmetabolism evident in diabetes, obesity, and cancer (1, 2). Owing to the fundamentalCopyright 2012 by the American Association for the Advancement of Science; all rights reserved.†To whom correspondence should be addressed. rutter@.*These authors contributed equally to this mentary /cgi/content/full/science.1218099/DC1

Bricker et -PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPage 2importance of pyruvate, the mitochondrial pyruvate carrier (MPC) has been studiedextensively (3, 4). This included the discovery that α-cyanocinnamate analogs, such asUK-5099, act as specific and potent inhibitors of carrier activity (5). In spite of thischaracterization, however, the gene or genes that encode the mitochondrial pyruvate carrierremain unknown (6, 7).As part of an ongoing effort to characterize mitochondrial proteins that are conservedthrough evolution, we initiated studies of the MPC protein family (originally designatedBRP44 and BRP44L in humans) (8). This family contains three members in

Saccharomycescerevisiae, encoded by

YGL080W,

YHR162W, and

YGR243W, hereafter referred to asMPC1,

MPC2, and

MPC3, respectively. Mpc2 and Mpc3 are 79% identical in amino acidsequence and appear to be the product of a recent gene duplication event. Mpc1, Mpc2, andMpc3 colocalize with mitochondria (Fig. 1A and fig. S2A), consistent with publishedmitochondrial proteomic studies (9, 10). The mitochondrial localization of Mpc1 and Mpc2was confirmed by biochemical fractionation (Fig. 1B). Mpc1, Mpc2, and Mpc3 wereenriched in mitochondrial membranes (fig. S2B), consistent with the presence of predictedtransmembrane domains in their sequences (fig. S1). Mpc1 and Mpc2 were resistant toprotease treatment unless the mitochondrial outer membrane was ruptured (Fig. 1B and fig.S2C), implying that they are embedded in the mitochondrial inner tographic purification of tagged variants of Mpc1 and Mpc2, followed by massspectrometry, revealed that Mpc2 and Mpc3 were among the major interacting proteins ofMpc1, and Mpc1 and Mpc3 were among the major interacting proteins of Mpc2 (table S1).Consistent with this, immunoprecipitation of tagged Mpc1 copurified Mpc2 and vice versa(Fig. 1C, lanes 3 and 4). In addition, Mpc2 can interact with itself (Fig. 1C, lane 8), whereasan Mpc1 homotypic interaction was not detected (Fig. 1C, lane 7). Blue native–polyacrylamide gel electrophoresis showed that both Mpc1 and Mpc2 migrated as part of an~150-kD complex (fig. S2D). Loss of Mpc2 prevented Mpc1 from migrating in thiscomplex, whereas an

mpc1Δ strain showed elevated Mpc2 complex formation (fig. S2E).We conclude that Mpc1 and Mpc2 form a multimeric complex embedded in themitochondrial inner membrane, with Mpc2 likely being the major structural yeast strains were subjected to a variety of growth conditions. The

mpc1Δ andmpc2Δ cells displayed mild growth defects on non-fermentable carbon sources likeglycerol, with greater effects on glucose medium (fig. S3) and a strong growth defect in theabsence of leucine (Fig. 1D). In contrast,

mpc3Δ mutant displayed no apparent growthphenotypes. Yeast,

Drosophila, or human

MPC1 orthologs, but not human

MPC2, couldrescue the

mpc1Δ growth phenotype (Fig. 1E), indicating that Mpc1 function is conservedthrough analyze the physiological function of MPCs in a multicellular animal, we extended ourstudies to the

Drosophila ortholog of MPC1 (dMPC1; encoded by

CG14290), which alsolocalized to mitochondria (fig. S4). Analogous to yeast

mpc1Δ mutants,

dMPC1 mutants(fig. S5) were viable on standard food, but sensitive to a carbohydrate-only diet, with rapidlethality after transfer to a sucrose medium (Fig. 2A). Whereas the amount of adenosine 5′-triphosphate (ATP) was reduced in

dMPC1 mutants (Fig. 2C), along with triacylglycerol(TAG) and protein (fig. S6, B and C), the amounts of carbohydrates were elevated,including the circulating sugar trehalose (Fig. 2D), glucose (Fig. 2E), fructose, and glycogen(fig. S6, A and D). These results suggest that

dMPC1 mutants are defective in carbohydratemetabolism and may consume stored fat and protein for energy. Consistent with this, thelethality of

dMPC1 mutants on the sugar diet was rescued by expression of the wild-typegene in tissues that depend heavily on glucose metabolism: the fat body, muscle, andneurons (Fig. 2B).Science. Author manuscript; available in PMC 2013 June 24.

Bricker et -PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPage 3Metabolomic analyses revealed that the concentration of pyruvate was highly elevated,whereas TCA cycle intermediates were significantly depleted in

dMPC1 mutants on thesugar diet (Fig. 2F). Similarly, the amounts of glycine and serine, which can interconvertwith glycolytic intermediates, were elevated in the mutants on the sugar diet (fig. S6E),whereas glutamate, aspartate, and proline, which can interconvert with TCA cycleintermediates, were depleted under these conditions (fig. S6F). Consistent with this,metabolomic analysis of

mpc1Δ and

mpc2Δ yeast mutants revealed elevated pyruvateconcentrations (Fig. 3A), depletion of malate (fig. S7), depleted acetyl–coenzyme A (CoA),and elevated CoA concentrations (Fig. 3B). Taken together, these results suggest that

MPC1mutants are unable to efficiently convert cytosolic pyruvate to mitochondrial acetyl-CoA todrive the TCA cycle and ATP phenotypes could arise from either a defect in mitochondrial pyruvate uptake or theconversion of mitochondrial pyruvate into acetyl-CoA by the pyruvate dehydrogenase(PDH) complex. Yeast lacking Mpc1, however, had nearly wild-type PDH activity, unlikethe strong decrease seen in

pda1Δ mutants (Fig. 3C), which lack PDH function (11). Adecrease in PDH activity also does not explain the growth defect of

mpc1Δ mutants, whichis more severe than that of the

pda1Δ mutant (fig. S8). However, combining the

mpc1Δallele with a deletion for

mae1, which encodes a malic enzyme that converts malate topyruvate in the mitochondrial matrix (12), revealed a profound growth defect on glucosemedium that was completely rescued by plasmid expression of either

MAE1 or

MPC114 (Fig.3D). Notably, mitochondria from the

mpc1Δ mutant displayed almost no uptake of C-pyruvate, which could be fully rescued by plasmid expression of wild-type

MPC1 (Fig. 3E).Moreover, Mpc1 appears to be a key target for UK-5099, which is an inhibitor of themitochondrial pyruvate carrier (5). The

mae1Δ

mpc1Δ double mutant displayed reducedgrowth on glucose medium lacking leucine, and this phenotype could be effectively rescuedby transgenic expression of wild-type

MPC1 in the absence, but not the presence, ofUK-5099 (Fig. 3F). By screening for

MPC1118 mutants that could grow in the presence ofUK-5099, we recovered an Asp→Gly (D118G) substitution in Mpc1 that conferredUK-5099 resistance (Fig. 3F). Moreover, whereas

14C-pyruvate uptake into mitochondriaexpressing wild-type

MPC1 was almost completely inhibited by UK-5099, efficientpyruvate uptake that is resistant to UK-5099 was recovered upon expression of

MPC1-D118G (Fig. 3G). We conclude that Mpc1 is a key component of the mitochondrial pyruvatecarrier that corresponds to the activity studied for decades by Halestrap and others (5, 13).Depletion of

MPC1 in mouse embryonic fibroblasts (fig. S9A)causeda modest decrease inpyruvate-driven oxygen consumption under basal conditions, and a stronger reduction in thepresence of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), which stimulatesmaximal respiration (Fig. 4A). Similar results were also seen upon silencing

MPC2 (Fig. 4Band fig. S9A). This suppression of pyruvate oxidation, which occurred without affectingcomponents of the oxidative phosphorylation machinery (fig. S9, B and C), suggests thatmammalian Mpc1 and Mpc2 mediate mitochondrial pyruvate uptake in a manner similar tothat seen in yeast and

have previously described a French-Algerian family with two offspring that exhibited adevastating defect in mitochondrial pyruvate oxidation (14) (Fig. 4C, family 1). Wesubsequently discovered two additional families, each with one affected child who displayeda similar, but less severe, phenotype (Fig. 4C, families 2 and 3). Linkage analysis andhomozygosity mapping allowed us to focus on one candidate region on chromosome 6(163,607,637 to 166,842,083, GRCh37/hg19). This interval contained 10 potential candidategenes:

PACRG,

QKI,

C6orf118,

PDE10A,

SDIM1,

T,

PRR18,

SFT2D1,

RPS6KA2, andBRP44L, which is the human

MPC1. DNA sequencing of the exons and intron/exonboundaries of the

MPC1 gene in fibroblasts from the affected patients in families 2 and 3Science. Author manuscript; available in PMC 2013 June 24.

Bricker et 4revealed the same molecular lesion, c.236T→A, causing a predicted 79→His (L79H)alteration (Fig. 4D). Analysis of DNA from family 1 revealed a distinct sequence change, c.289C→T, which resulted in a predicted 97→Trp (R97W) mutation (Fig. 4D). Both ofthe affected residues are conserved through evolution between

MPC1 orthologs, and Arg97is conserved among both

MPC1 and

MPC2 orthologs (fig. S1).Cells from the affected individuals in families 1 and 2 exhibited impaired basal and FCCP-stimulated pyruvate oxidation (Fig. 4E), whereas glutamine-driven oxygen consumption wasnormal or elevated, demonstrating that they have not acquired a generalized impairment ofmitochondrial respiration (Fig. 4E). As expected, expression of wild-type human

MPC1 inthe cells from family 2 (Fig. 4F) or family 1 (Fig. 4G) either completely or partially rescuedthe defect in FCCP-induced pyruvate oxidation. Moreover, expression of the

MPC1-Leu79His allele was less effective at suppressing the yeast

mpc1Δ growth defect relative towild-type human

MPC1 (Fig. 4H), and the stronger

MPC1-Arg97Trp allele was essentiallyinactive (Fig. 4H), suggesting that

MPC1 function is evolutionarily conserved from yeast data presented here demonstrate that the Mpc1-Mpc2 complex is an essentialcomponent of the mitochondrial pyruvate carrier in yeast, flies, and mammals. This isconsistent with experiments performed in rat liver, heart, and castor beans, which implicatedproteins of 12 to 15 kD in mitochondrial pyruvate uptake (15)—similar to the molecularmasses of Mpc1 (15 kD), Mpc2 (14 kD), and Mpc3 (16 kD). Although these individual sizesare relatively small, Mpc1 and Mpc2 form a complex of ~150 kD, suggesting that anoligomeric structure mediates pyruvate transport. The demonstration that Mpc1 and Mpc2are sufficient to promote pyruvate uptake in a heterologous system provides further evidencethat they constitute an essential pyruvate transporter (16). Finally, the degree to whichcarbohydrates are imported into mitochondria and converted into acetyl-CoA is a criticalstep in normal glucose oxidation as well as the onset of diabetes, obesity, and cancer. Thus,like PDH, which is controlled by allostery and posttranslational modification (17), themitochondrial import of pyruvate is likely to be precisely regulated (18, 19). Theidentification of Mpc1 and Mpc2 as critical for mitochondrial pyruvate transport provides anew framework for understanding this level of metabolic control, as well as new directionsfor potential therapeutic -PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptSupplementary MaterialRefer to Web version on PubMed Central for supplementary ledgmentsWe thank members of the Rutter, Thummel, Winge, Stillman, Shaw, and Metzstein laboratories for helpfuldiscussions. We thank the Shaw and Winge labs for the antibodies against Fzo1, Cyb2, and Mge1 and for the mito-RFP constructs. We thank J. M. Saudubray, L. Burglen, and H. Tevissen for referring patients and C. Thibault andJ. L. Mandel (IGBMC, Strasbourg, France) for assistance in single-nucleotide polymorphism array research was supported by NIH grants R01GM083746 (J.R.), RC1DK086426 (C.S.T.), and R24DK092784(J.R. and C.S.T.) and a pilot grant from P30DK072437 (J.R.). D.K.B. and C.M.C. were supported by the NIHGenetics Predoctoral Training Grant T32GM007464. E.B.T. was supported by NIH Pathway to Independenceaward K99AR059190. D.K.B., T.O., C.S.T., and J.R. are inventors on a patent application by the University ofUtah covering the discovery of the MPC nces and Notes1. Hanahan D, Weinberg RA. Cell. 2011; 144:646. [PubMed: 21376230]2. Kahn SE, Hull RL, Utzschneider KM. Nature. 2006; 444:840. [PubMed: 17167471]3. Halestrap AP, Denton RM. Biochem J. 1974; 138:313. [PubMed: 4822737]Science. Author manuscript; available in PMC 2013 June 24.

Bricker et -PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPage 54. Halestrap AP. Biochem J. 1978; 172:377. [PubMed: 28726]5. Halestrap AP. Biochem J. 1975; 148:85. [PubMed: 1156402]6. Todisco S, Agrimi G, Castegna A, Palmieri F. J Biol Chem. 2006; 281:1524. [PubMed: 16291748]7. Hildyard JC, Halestrap AP. Biochem J. 2003; 374:607. [PubMed: 12887330]8. Jiang M, et al. Mol Biol Rep. 2009; 36:215. [PubMed: 18026869]9. Pagliarini DJ, et al. Cell. 2008; 134:112. [PubMed: 18614015]10. Sickmann A, et al. Proc Natl Acad Sci USA. 2003; 100:13207. [PubMed: 14576278]11. Steensma HY, Holterman L, Dekker I, van Sluis CA, Wenzel TJ. Eur J Biochem. 1990; 191:769.[PubMed: 2202601]12. Boles E, de Jong-Gubbels P, Pronk JT. J Bacteriol. 1998; 180:2875. [PubMed: 9603875]13. Papa S, Paradies G. Eur J Biochem. 1974; 49:265. [PubMed: 4459142]14. Brivet M, et al. Mol Genet Metab. 2003; 78:186. [PubMed: 12649063]15. Thomas AP, Halestrap AP. Biochem J. 1981; 196:471. [PubMed: 7316989]16. Herzig S, et al. Science. 2012; 337:93. [PubMed: 22628554]17. Harris RA, Bowker-Kinley MM, Huang B, Wu P. Adv Enzyme Regul. 2002; 42:249. [PubMed:12123719]18. Zwiebel FM, Schwabe U, Olson MS, Scholz R. Biochemistry. 1982; 21:346. [PubMed: 7074018]19. Rognstad R. Int J Biochem. 1983; 15:1417. [PubMed: 6653863]20. Materials and methods are available as supplementary materials on

Science e. Author manuscript; available in PMC 2013 June 24.

Bricker et -PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPage 6Fig. 1 and Mpc2 are evolutionarily conserved mitochondrial inner-membrane proteins. (A)Mpc1 labeled with green fluorescent protein (Mpc1-GFP) and mitochondrial targeted redfluorescent protein (MtRFP) coexpressed in yeast cells. DIC, differential interferencecontrast. (B) Intact mitochondria, hypotonic-swollen mitoplasts, and TritonX-100–solubilized mitochondria from a strain expressing Mpc1-V5 and Mpc2-His6/HA2 with (+) orwithout (−) proteinase K incubation. An immunoblot of extracts using the indicatedantibodies with the whole-cell lysate (WCL) and postmitochondrial supernatant (PMS) isshown. Mge1, Cyb2, and Fzo1 are matrix, intermembrane space, and outer-membraneproteins, respectively. (C) Immunoprecipitations from mitochondrial extracts from

mpc1Δmpc2Δ cells expressing Mpc1 and Mpc2 tagged as indicated. Immunoblot of eitherimmunoprecipitate (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 leucineand grown at 30°C for 24 hours. (E) Serial dilutions of indicated strains spotted on syntheticmedia lacking leucine and grown at 30°C for 48 hours.

wt, wild type; EV, empty e. Author manuscript; available in PMC 2013 June 24.

Bricker et -PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPage 7Fig. 1 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) Relativeabundance of pyruvate and TCA cycle intermediates in

dMPC1 or

dMPC− flies after 2 dayson 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 ± e. Author manuscript; available in PMC 2013 June 24.

Bricker et 8NIH-PA Author ManuscriptFig. -PA Author ManuscriptNIH-PA Author ManuscriptMPC1 is required for mitochondrial pyruvate uptake. (A) Relative abundance of pyruvate inthe indicated strains.

P values relative to

wt. (B) Relative abundance of acetyl-CoA and CoAin the indicated strains. (C) Mitochondrial pyruvate dehydrogenase activity in the indicatedstrains.

P value relative to

wt and

mpc1Δ. (D) Serial dilutions of the indicated strain onglucose medium grown at 30°C for 48 hours. (E) Uptake of

14C-pyruvate into mitochondriapurified from either

wt or

mpc1Δ cells containing the indicated plasmid.

P value relative towt + EV and

mpc1Δ +

MPC1. (F)

Mae1Δ

mpc1Δ cells transformed with the indicatedplasmid 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 theindicated 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 ± e. Author manuscript; available in PMC 2013 June 24.

Bricker et -PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPage 9Fig. ian

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).

Pvalues 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, mark individuals from whom fibroblasts were obtained. (D) The protein region ofMPC1 containing the predicted amino acid substitutions from all three families aligned byClustalW. 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, Arabidopsisthaliana. (E) Pyruvate- (left) and glutamine- (right) supported respiration of fibroblastsharboring either L79H or R97W

MPC1 mutations. (F) Pyruvate-supported respiration ofeither 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 aftertransduction with the indicated vector. (H) Serial dilutions of

wt or

mae1Δ

mpc1Δ yeaststrains carrying the indicated plasmid grown on medium lacking uracil for plasmid selectionat 30°C for 40 hours. Both long (L) and short (S) forms of R97W were used (with or withoutexon 4). ***P < 0.001, **P < 0.01, *P < 0.05, †P < 0.10; NS, not significant (Student’s

ttest). Data are shown as mean ± e. Author manuscript; available in PMC 2013 June 24.