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Identification and Functional Expression of the Mitochondrial Pyruvate Carrier

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Science  06 Jul 2012:
Vol. 337, Issue 6090, pp. 93-96
DOI: 10.1126/science.1218530

Abstract

The transport of pyruvate, the end product of glycolysis, into mitochondria is an essential process that provides the organelle with a major oxidative fuel. Although the existence of a specific mitochondrial pyruvate carrier (MPC) has been anticipated, its molecular identity remained unknown. We report that MPC is a heterocomplex formed by two members of a family of previously uncharacterized membrane proteins that are conserved from yeast to mammals. Members of the MPC family were found in the inner mitochondrial membrane, and yeast mutants lacking MPC proteins showed severe defects in mitochondrial pyruvate uptake. Coexpression of mouse MPC1 and MPC2 in Lactococcus lactis promoted transport of pyruvate across the membrane. These observations firmly establish these proteins as essential components of the MPC.

In the mitochondrion, pyruvate is converted into acetyl–coenzyme A (acetyl-CoA) by the pyruvate dehydrogenase (PDH) complex and participates in the synthesis of branched-chain amino acids (BCAAs) in yeast. Acetyl-CoA donates carbon atoms to the citric acid cycle and participates in the synthesis of octanoic acid, the precursor of lipoic acid (1). Lipoic acid is an essential cofactor of several multi-subunit complexes in the mitochondrial matrix, including PDH, α-ketoglutarate dehydrogenase (α-KDH), and the branched-chain keto acid dehydrogenase (BCKDH) (2). Import of pyruvate across the inner mitochondrial membrane (IMM) requires a specific carrier whose molecular identity has not been established (3). We have identified a family of membrane proteins (pfam UPF0041) whose members are necessary and sufficient for the transport of pyruvate into mitochondria. We have renamed this family the mitochondrial pyruvate carrier (MPC) family.

We previously identified MPC1 (formerly Brp44L) in a proteomic analysis of the IMM of mouse liver (4). MPC1 and its paralog MPC2 (formerly Brp44) have unknown function. Both are IMM proteins with three predicted transmembrane α-helices on the basis of secondary structure predictions and hydropathy profiling (fig. S1). They share sequence similarity with yeast Mpc1 (Ygl080w), Mpc2 (Yhr162w), and Mpc3 (Ygr243w) (fig. S1, C and D). We used Saccharomyces cerevisiae as a model organism to investigate the function of this family of proteins.

In yeast, Mpc1 localized in the IMM (fig. S2). Growth of the knockout mutants mpc1Δ, mpc2Δ, mpc3Δ, and mpc2Δmpc3Δ was normal on rich medium, either containing fermentable [yeast extract, peptone, and dextrose (YPD)] or nonfermentable carbon sources [Yeast extract, peptone, glycerol (YPG)] (Fig. 1A). In contrast, mpc1Δ and mpc2Δ mpc3Δ cells grew more slowly in amino acid–free medium [synthetic dextrose (SD)] than did the cells of the isogenic wild-type (WT) strain (Fig. 1A and fig. S3A). Deletion of MPC2 alone led to a minor growth defect, whereas deletion of MPC3 had no visible effect on growth. A similar phenotype was observed by (5).

Fig. 1

Phenotypes of yeast deleted for MPC genes. (A) Spot assays of strains (as indicated) on media differing in amino acid composition or carbon source. The plates are representative of at least three independent experiments. (B) Spot assays of mpc1Δ cells transformed with p2U vector containing the indicated gene. (C) Spot assays of mpc2Δmpc3Δ cells transformed with p2U vector containing the indicated gene. (D) Valine or leucine decarboxylation in WT (black bars) or mpc1Δ (white bars) cells. The means of six independent experiments are indicated above the bars ± SD. (E) Valine decarboxylation in the indicated strains. The mean of at least three independent experiments ± SD is shown. (F) PDH and α-KDH activities in WT (black bars) or mpc1Δ (white bars) mitochondria. Mean ± SD of three independent experiments for PDH and two independent experiments for α-KDH are shown.

Expression of WT MPC1 restored growth of mpc1Δ cells (Fig. 1, B and C). Suppressor screens for genes that restored growth of mpc1Δ cells in SD identified only MPC1 (supplementary text), suggesting that its function cannot be complemented by other genes. Expression of either MPC2 or MPC3 restored growth of mpc2Δmpc3Δ cells but not that of cells lacking MPC1 (Fig. 1, B and C). Thus, the growth of yeast in SD required a combination of Mpc1 with Mpc2 or Mpc3. We found that Mpc1 coimmunoprecipitated with Mpc2, suggesting that they form a heterocomplex (fig. S3B).

The growth defect in SD was relieved by addition of valine or leucine, with an additive effect of both amino acids (Fig. 1A). In contrast, addition of all amino acids except leucine and valine did not restore growth of the mutant strains (SC –V –L) (Fig. 1A). This finding prompted us to investigate the role of MPC proteins in the metabolism of valine and leucine. We assayed their decarboxylation by monitoring the release of 14CO2 by cells grown in SD supplemented with either 1-14C valine or 1-14C leucine. Release of 14CO2 by mpc1Δ cells was less than 2% of that in WT cells (Fig. 1D). A similar defect occurs in a lpd1Δ strain (Fig. 1E) (6), lacking a lipoamide dehydrogenase essential for the function of the mitochondrial dehydrogenase complexes, PDH, α-KDH (7), and BCKDH (8). To test whether mpc1Δ cells also had dysfunctional PDH and α-KDH, we assessed their activities in mitochondria. The activity of these two complexes was impaired in mitochondria from mpc1Δ cells grown in SD (Fig. 1F), suggesting that the function of Mpc1 extends to several lipoyl-dependent complexes.

Lipoic acid is covalently attached to the E2 subunits of PDH (Lat1) and α-KDH (Kgd2), the E3-binding protein of PDH (Pdx1), and the H subunit of the glycine cleavage system (Gcv3) (9, 10). This modification can be readily assessed by means of protein immunoblotting with antibodies to lipoic acid (11). In rich medium, WT, mpc1Δ, mpc2Δ, mpc3Δ, and mpc2Δmpc3Δ cells had similar amounts of lipoylated complexes (Fig. 2A). However, when cells were grown in SD, lipoylated proteins were virtually absent from mpc1Δ, mpc2Δ, and mpc2Δmpc3Δ but not mpc3Δ cell lysates (Fig. 2A). Addition of valine or leucine, or both, to SD or expression of MPC1 restored lipoylation in mpc1Δ (Fig. 2B and fig. S3C). The defect in lipoylation was correlated with decreased abundance of lipoic acid in mpc1Δ cells grown in SD (fig. S3D). In contrast, lipoic acid was abundant in mpc1Δ cells grown in rich medium (fig. S3D). Thus, Mpc1 is essential for the production of lipoic acid and the function of lipoyl-dependent complexes only when cells are grown in SD, and this defect is relieved by the addition of valine and leucine.

Fig. 2

Lipoic acid production defects in mpc mutant strains (A and B) Protein immunoblot analysis of cell extracts using antibodies raised against lipoic acid in strains and media as indicated. Ponceau staining is shown as loading control. The blots are representative of at least three independent experiments. (C) PDH activity of mitochondria isolated from the indicated strains grown in lactate medium. N.D., not detected. Data are expressed as the mean ± SD of three independent experiments.

We tested at which level the pathway leading to lipoic acid synthesis was defective in mpc1Δ cells grown in SD (fig. S4) (12). We excluded defects in the mitochondrial acetyl-CoA carboxylase HFA1, which converts acetyl-CoA into malonyl-CoA, or in the lipoic acid synthase Lip5, which converts octanoic acid into lipoic acid because cells lacking these enzymes cannot produce lipoic acid even when grown in rich medium (9, 13). In addition, no defect in the synthesis of octanoic acid from malonyl-CoA was detected in mitochondria isolated from mpc1Δ cells grown in SD medium (fig. S3E). Thus, the function of Mpc1 was likely to be upstream of acetyl-CoA (fig. S4). Because it is impossible to accurately measure amounts of acetyl-CoA in mitochondria, we tested the impact of reduced amount of mitochondrial acetyl-CoA on protein lipoylation by inhibiting PDH, the main source of acetyl-CoA from pyruvate. PDH-deficient cells (pdx1Δ) (10) were defective in protein lipoylation in SD, which was restored by addition of valine and leucine, similarly to mpc1Δ cells (Fig. 2B). However, PDH activity was almost normal in mpc1Δ cells grown in rich medium [when the E2 subunit was lipoylated (Fig. 2C)]. Thus, the MPC proteins appeared to act upstream of PDH and may function in the transport of pyruvate into mitochondria. We therefore measured uptake of 14C pyruvate in mitochondria isolated from WT, mpc1Δ, mpc2Δ, mpc3Δ, and mpc2Δmpc3Δ cells grown in lactate medium (Fig. 3A). The specificity of uptake was assessed by the use of UK5099, an inhibitor of the mitochondrial pyruvate carrier (14). Uptake of pyruvate in WT mitochondria was sensitive to the proton ionophore carbonyl cyanide m-chloro phenyl hydrazone (CCCP) (Fig. 3B). Mitochondria from mpc1Δ and mpc2Δmpc3Δ cells showed decreased pyruvate uptake (Fig. 3, A and B), despite a normal mitochondrial membrane potential (fig. S5). Surprisingly, deletion of MPC3 alone impaired pyruvate uptake in mitochondria, whereas mitochondria from the mpc2Δ mutant transported pyruvate normally. Because this result did not correlate with the phenotypes of mpc2Δ and mpc3Δ single mutants grown in SD, we investigated the expression of Mpc2 and Mpc3 in SD and lactate media. In SD, yeast expressed mainly Mpc2, whereas in lactate medium, they mainly expressed Mpc3 (Fig. 3C). This expression pattern could be explained, at least in part, by the presence of binding sites for Gcn4 (a transcription factor activated by amino acid starvation) upstream of MPC2 (15). This raises the possibility that under certain growth conditions, these two proteins might have specific, nonredundant functions.

Fig. 3

Impaired import of pyruvate in mpc mutant strains. (A) Kinetics of pyruvate import into isolated mitochondria of WT (closed circles), mpc1Δ (open circles), mpc2Δ (closed triangles), mpc3Δ (open triangles), or mpc2Δmpc3Δ (squares) cells. Data are representative of at least three independent experiments. (B) Rate of pyruvate import calculated from the first 2 minutes of import. Data are mean of at least three independent experiments ± SEM. N.D., not detected. (C) Protein immunoblot analysis of protein levels using genomically 3× hemagglutinin (HA)–tagged proteins in the indicated medium, probed with antibodies to HA. Ponceau staining is shown as a loading control. The blot is representative of three independent experiments.

We next assessed whether mouse MPC1 (mMPC1) and MPC2 (mMPC2) could restore growth of yeast cells lacking a functional pyruvate transporter (Fig. 4, A and B). mMPC1 alone restored growth of mpc1Δ cells, but mMPC2 failed to restore growth of the double-deletion strain of its orthologous genes MPC2 and MPC3. However, growth of the triple-deletion strain mpc1Δmpc2Δmpc3Δ or of mpc2Δmpc3Δ cells was restored by coexpression of both mMPC1 and mMPC2 (Fig. 4A). Thus, mMPC1 and mMPC2 together functionally complement the absence of pyruvate transport. We next expressed mMPC1 and mMPC2, alone and in combination, in the bacterium Lactococcus lactis (Fig. 4C), which has been successfully used to express and characterize mitochondrial transporters (16). No pyruvate uptake was observed in bacteria expressing either protein alone compared with the empty vector control. However, a fourfold increase in pyruvate uptake was detected when mMPC1 and mMPC2 were coexpressed (Fig. 4, D and E). This uptake was sensitive to the mitochondrial pyruvate carrier inhibitor UK5099 and to 2-deoxyglucose, which collapses the proton electrochemical gradient (Fig. 4E) (17). Moreover, artificially increasing the membrane potential by lowering the pH in the import buffer from 7.2 to 6.2 significantly increased pyruvate uptake (two-tailed t test, P < 0.05) (Fig. 4E). Thus, coexpression of mMPC1 and mMPC2 in bacteria is sufficient to allow import of pyruvate with similar properties to the mitochondrial pyruvate carrier (3). We therefore conclude that the mitochondrial pyruvate carrier is composed of Mpc1 and either Mpc2 or Mpc3 in yeast and of MPC1 and MPC2 in mammals.

Fig. 4

mMPC1 and mMPC2 functionally reconstitute the pyruvate carrier in L. lactis. (A) Spot assay in amino acid–free medium of the indicated yeast strains transformed with an empty vector or expressing mMPC1 or mMPC2. (B) Protein immunoblot analysis of expression of the different proteins from strains shown in (A). (C) Protein immunoblotting of L. lactis strains showing expression of mMPC proteins. (D) Time course of pyruvate import in L. lactis strains with empty vector (diamonds), mMPC1 (circles), mMPC2 (triangles), or both (squares). The mean of three independent experiment ± SEM is shown. (E) Quantification of the import rate from the first 20 min of import in L. lactis strains expressing the indicated proteins. UK 5099 was at 50 μM. 2DG, 30 mM 2-deoxyglucose. *P < 0.05, two-tailed t test.

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1218530/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S5

Table S1

References (1823)

References and Notes

  1. Acknowledgments: We are grateful to R. Loewith and F. Stutz for strains and technical help, L. Szweda for antibodies, A. Kastaniotis for technical help on lipoic acid determination, Y. Que for erythromycin resistance cassette, and H. Riezman, A. Jourdain, and the Martinou lab for fruitful discussions. This work was supported by Novartis Science Foundation (S.H.), the Swiss National Science Foundation (subsidy 31003A-141068/1 to J.-C.M.), and the state of Geneva.
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