biochem;

biochem; to the questions using a maximum of ~500 words. Note that all DropBox files are automatically evaluated by turnitin.com for plagiarism against all web and class document s . DO NOT INCLUDE THE QUESTIONS IN YOUR DOCUMENT – ONLY THE ANSWERS – AND DO NOT QUOTE DIRECTLY FROM THE PAPER. You need to use your own words. Read the attached article: “ A Mitochondrial Pyruvate Carrier Required for Py ruvate Uptake in Yeast, Drosophila, and Humans,” Science , 337 , p. 96 - 100 and answer the following questions. The authors describe experiments performed to identify the carrier protein involved in pyruvate uptake into the mitochondria. 1) How did the authors demonstrate that the Mpc1 protein was located in the mitochondrial inner membrane? 2) Describe how the authors demonstrate d that Mpc1 protein function was conserved through evolution. 3) The Drosophila that lacked dMPC1 (dMPC1 - ) died q uickly on a sugar only diet (Figure 2A). What does this suggest about sugar metabolism in these flies? How did measurement of metabolites in Figure 2F, 3A and 3B help the authors understand why the dMPC1 - flies were dying? 4) Even without a pyruvate carri er to transport pyruvate from the cytosol to the matrix, the mitochondri a can still have some pyruvate in the matrix. Describe one way that pyruvate can be generated in the mitochondrial matrix in the absence of Mpc1. 5) Describe the experiment in this pa per that most directly demonstrates that Mpc1 is a pyruvate carrier. PDH activity was almost normal in mpc1 D cells grown in rich medium [when the E2 subunit was lipoylated (Fig. 2C)]. Thus, the MPC proteins ap- peared to act upstream of PDH and may function in the transport of pyruvate into mitochondria. We therefore measured uptake of 14 C pyruvate in mitochondria isolated from WT, mpc1 D , mpc2 D , mpc3 D ,and mpc2 D mpc3 D 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). Mitochon- dria from mpc1 D and mpc2 D mpc3 D cells showed decreased pyruvate uptake (Fig. 3, A and B), de- spite a normal mitochondrial membrane poten- tial (fig. S5). Surprisingly, deletion of MPC3 alone impaired pyruvate uptake in mitochondria, where- as mitochondria from the mpc2 D mutant trans- ported pyruvate normally. Because this result did not correlate with the phenotypes of mpc2 D and mpc3 D single mutants grown in SD, we inves- tigated 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 pat- tern could be explained, at least in part, by the presence of binding sites for Gcn4 (a transcrip- tion factor activated by amino acid starvation) upstream of MPC2 ( 15 ). This raises the possi- bility that under certain growth conditions, these two proteins might have specific, nonredundant functions. We next assessed whether mouse MPC1 (mMPC1) and MPC2 (mMPC2) could restore growth of yeast cells lacking a functional pyru- vate transporter (Fig. 4, A and B). mMPC1 alone restored growth of mpc1 D 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 mpc1 D mpc2 D mpc3 D or of mpc2 D mpc3 D 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 ex- pressing either protein alone compared with the empty vector control. However, a fourfold in- crease in pyruvate uptake was detected when mMPC1 and mMPC2 were coexpressed (Fig. 4, D and E). This uptake was sensitive to the mito- chondrial pyruvate carrier inhibitor UK5099 and to 2-deoxyglucose, which collapses the proton elec- trochemical 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, coex- pression 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. References and Notes 1. J. K. Hiltunen, Z. Chen, A. M. Haapalainen, R. K. Wierenga, A. J. Kastaniotis, Prog. Lipid Res. 49 , 27 (2010). 2. L. J. Reed, J. Biol. Chem. 276 , 38329 (2001). 3. A. P. Halestrap, Biochem. J. 148 , 85 (1975). 4. S. Da Cruz et al ., J. Biol. Chem. 278 , 41566 (2003). 5. D. K. Bricker et al ., Science 337 , 96 (2012). 6. J. R. Dickinson, I. W. Dawes, J. Gen. Microbiol. 138 , 2029 (1992). 7. J. R. Dickinson, D. J. Roy, I. W. Dawes, Mol. Gen. Genet. 204 , 103 (1986). 8. R. A. Harris, M. Joshi, N. H. Jeoung, M. Obayashi, J. Nutr. 135 (suppl.), 1527S (2005). 9. M. S. Schonauer, A. J. Kastaniotis, V. A. Kursu, J. K. Hiltunen, C. L. Dieckmann, J. Biol. Chem. 284 , 23234 (2009). 10. J. E. Lawson, R. H. Behal, L. J. Reed, Biochemistry 30 , 2834 (1991). 11. K. M. Humphries, L. I. Szweda, Biochemistry 37 , 15835 (1998). 12. H. Wada, D. Shintani, J. Ohlrogge, Proc. Natl. Acad. Sci. U.S.A. 94 , 1591 (1997). 13. U. Hoja et al ., J. Biol. Chem. 279 , 21779 (2004). 14. A. P. Halestrap, R. M. Denton, Biochem. J. 148 ,97 (1975). 15. K. D. MacIsaac et al ., BMC Bioinformatics 7 , 113 (2006). 16. E. R. Kunji, D. J. Slotboom, B. Poolman, Biochim. Biophys. Acta 1610 , 97 (2003). 17. E. R. Kunji, E. J. Smid, R. Plapp, B. Poolman, W. N. Konings, J. Bacteriol. 175 , 2052 (1993). 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. Supplementary Materials www.sciencemag.org/cgi/content/full/science.1218530/DC1 Materials and Methods Supplementary Text Figs. S1 to S5 Table S1 References ( 18 – 23 ) 29 December 2011; accepted 11 May 2012 Published online 24 May 2012; 10.1126/science.1218530 A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila , and Humans Daniel K. Bricker, 1 * Eric B. Taylor, 2 * John C. Schell, 2 * Thomas Orsak, 2 * Audrey Boutron, 3 Yu-Chan Chen, 2 James E. Cox, 4 Caleb M. Cardon, 2 Jonathan G. Van Vranken, 2 Noah Dephoure, 5 Claire Redin, 6 Sihem Boudina, 7 Steven P. Gygi, 5 Michèle Brivet, 3 Carl S. Thummel, 1 Jared Rutter 2 † 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. P yruvate occupies a pivotal node in the reg- ulation of carbon metabolism as it is the end product of glycolysis and a major sub- strate for the tricarboxylic acid (TCA) cycle in mitochondria. Pyruvate lies at the intersection of these catabolic pathways with anabolic path- ways for lipid synthesis, amino acid biosynthesis, and gluconeogenesis. As a result, the failure to correctly partition carbon between these fates lies at the heart of the altered metabolism evi- dent in diabetes, obesity, and cancer ( 1 , 2 ). Owing to the fundamental importance of pyruvate, the mitochondrial pyruvate carrier (MPC) has been studied extensively ( 3 , 4 ). This included the dis- covery that a -cyanocinnamate analogs, such as UK-5099, act as specific and potent inhibitors of carrier activity ( 5 ). In spite of this character- ization, however, the gene or genes that encode the mitochondrial pyruvate carrier remain un- known ( 6 , 7 ). As part of an ongoing effort to characterize mitochondrial proteins that are conserved through evolution, we initiated studies of the MPC protein family (originally designated BRP44 and BRP44L 6 JULY 2012 VOL 337 SCIENCE www.sciencemag.org 96 REPORTS on November 5, 2012 www.sciencemag.org Downloaded from PLACE THIS ORDER OR A SIMILAR ORDER WITH US TODAY AND GET AN AMAZING DISCOUNT :)