Antonie van Leeuwenhoek

, Volume 102, Issue 1, pp 163–175

Mitochondrial involvement to methylglyoxal detoxification: d-Lactate/Malate antiporter in Saccharomyces cerevisiae

Original Paper


Research during the last years has accumulated a large body of data that suggest that a permanent high flux through the glycolytic pathway may be a source of intracellular toxicity via continuous generation of endogenous reactive dicarbonyl compound methylglyoxal (MG). MG detoxification by the action of the glyoxalase system produces d-lactate. Thus, this article extends our previous work and presents new insights concerning d-lactate fate in aerobically grown yeast cells. Biochemical studies using intact functional mitochondrial preparations derived from Saccharomyces cerevisiae show that d-lactate produced in the extramitochondrial phase can be taken up by mitochondria, metabolised inside the organelles with efflux of newly synthesized malate. Experiments were carried out photometrically and the rate of malate efflux was measured by use of NADP+ and malic enzyme and it depended on the rate of transport across the mitochondrial membrane. It showed saturation characteristics (Km = 20 μM; Vmax = 6 nmol min−1 mg−1 of mitochondrial protein) and was inhibited by α-cyanocinnamate, a non-penetrant compound. Our data reveal that reducing equivalents export from mitochondria is due to the occurrence of a putative d-lactate/malate antiporter which differs from both d-lactate/pyruvate antiporter and d-lactate/H+ symporter as shown by the different Vmax values, pH profile and inhibitor sensitivity. Based on these results we propose that d-lactate translocators and d-lactate dehydrogenases work together for decreasing the production of MG from the cytosol, thus mitochondria could play a pro-survival role in the metabolic stress response as well as for d-lactate-dependent gluconeogenesis.


Saccharomyces cerevisiae mitochondria d-Lactate metabolism Malate transport Methylglyoxal Reactive dicarbonyl compound Gluconeogenesis 



Saccharomyces cerevisiae mitochondria






Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone


Membrane potential


  1. Aguilera J, Prieto JA (2001) The Saccharomyces cerevisiae aldose reductase is implied in the metabolism of methylglyoxal in response to stress conditions. Curr Genet 39(5–6):273–283PubMedCrossRefGoogle Scholar
  2. Aguilera J, Prieto JA (2004) Yeast cells display a regulatory mechanism in response to methylglyoxal. FEMS Yeast Res 4(6):633–641PubMedCrossRefGoogle Scholar
  3. Bito A, Haider M, Hadler I, Breitenbach M (1997) Identification, phenotypic analysis of two glyoxalase II encoding genes from Saccharomyces cerevisiae, GLO2 and GLO4, and intracellular localization of the corresponding proteins. J Biol Chem 272:21509–21519PubMedCrossRefGoogle Scholar
  4. Bito A, Haider M, Briza P, Strasser P, Breitenbach M (1999) Heterologous expression, purification, and kinetic comparison of the cytoplasmic and mitochondrial glyoxalase II enzymes, Glo2p and Glo4p, from Saccharomyces cerevisiae. Protein Exp Purif 17:456–464CrossRefGoogle Scholar
  5. Boles E, de Jong-Gubbels P, Pronk JT (1998) Identification and characterization of MAE1, the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme. J Bacteriol 180:2875–2882PubMedGoogle Scholar
  6. Castegna A, Scarcia P, Agrimi G, Palmieri L, Rottensteiner H, Spera I, Germinario L, Palmieri F (2010) Identification and functional characterization of a novel mitochondrial carrier for citrate and oxoglutarate in Saccharomyces cerevisiae. J Biol Chem 285:17359–17370PubMedCrossRefGoogle Scholar
  7. Chambers P, Issaka A, Palecek SP (2004) Saccharomyces cerevisiae JEN1 promoter activity is inversely related to concentration of repressing sugar. Appl Environ Microbiol 70:8–17PubMedCrossRefGoogle Scholar
  8. Chelstowska A, Liu Z, Jia Y, Amberg D, Butow RA (1999) Signalling between mitochondria and the nucleus regulates the expression of a new d-lactate dehydrogenase activity in yeast. Yeast 15:1377–1391PubMedCrossRefGoogle Scholar
  9. Diaz-Ruiz R, Uribe-Carvajal S, Devin A, Rigoulet M (2009) Tumor cell energy metabolism and its common features with yeast metabolism. Biochim Biophys Acta 1796:252–265PubMedCrossRefGoogle Scholar
  10. Di Martino C, Pallotta ML (2011) Mitochondria-localized NAD biosynthesis by nicotinamide mononucleotide adenylyltransferase in Jerusalem artichoke (Helianthus tuberosus L.) heterotrophic tissues. Planta 234(4):657–7010PubMedCrossRefGoogle Scholar
  11. Di Martino C, Pizzuto R, Pallotta ML, De Santis A, Passarella S (2006) Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings. Planta 223:1123–1133PubMedCrossRefGoogle Scholar
  12. Dixon M (1953) The determination of enzyme inhibitor constants. Biochem J 55:170–171PubMedGoogle Scholar
  13. Douce R, Bourguignon J, Brouquisse R, Neuburger M (1987) Isolation of plant mitochondria: general principles and criteria of integrity. Methods Enzymol 148:403–415CrossRefGoogle Scholar
  14. Estojak J, Brent R, Golemis EA (1995) Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol 15:5820–5829PubMedGoogle Scholar
  15. Fratianni A, Pastore D, Pallotta ML, Chiatante D, Passarella S (2001) Increase of membrane permeability of mitochondria isolated from water stress adapted potato cells. Biosci Rep 21:81–91PubMedCrossRefGoogle Scholar
  16. Frazier AE, Chacinska A, Truscott AN, Guiard B, Pfanner N, Rehling P (2003) Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space. Mol Cell Biol 23:7818–7828PubMedCrossRefGoogle Scholar
  17. Gibson N, McAlister-Henn L (2003) Physical and genetic interactions of cytosolic malate dehydrogenase with other gluconeogenic enzymes. J Biol Chem 278(28):25628–25636PubMedCrossRefGoogle Scholar
  18. Gomes RA, Silva MS, Miranda HV, Ferreira AEN, Cordeiro CAA, Freire AP (2005) Protein glycation in Saccharomyces cerevisiae Argpyrimidine formation and methylglyoxal catabolism. FEBS J 272:4521–4531PubMedCrossRefGoogle Scholar
  19. Gomes RA, Vicente Miranda H, Silva MS, Graça G, Coelho AV, Ferreira AE, Cordeiro C, Freire AP (2006) Yeast protein glycation in vivo by methylglyoxal. Molecular modification of glycolytic enzymes and heat shock proteins. FEBS J 273(23):5273–5287PubMedCrossRefGoogle Scholar
  20. Hachiya NS, Sakasegawa Y, Jozuka A, Tsukita S, Kaneko K (2004) Interaction of d-lactate dehydrogenase protein 2 (Dld2p) with F-actin: implication for an alternative function of Dld2p. Biochem Biophys Res Commun 319(1):78–82PubMedCrossRefGoogle Scholar
  21. Halestrap AP, Denton RM (1975) The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds. Biochem J 148:97–106PubMedGoogle Scholar
  22. He W, Miao FJ, Lin DC, Schwandner RT, Wang Z, Gao Chen JL, Tian H, Ling L (2004) Citric acid cycle intermediates are ligands for orphan G-protein-coupled receptors. Nature 429:188–193PubMedCrossRefGoogle Scholar
  23. Hipkiss AR (2006) Dietary restriction, glycolysis, hormesis and ageing. Biogerontology 8:221–224PubMedCrossRefGoogle Scholar
  24. Hipkiss AR (2009) NAD+ availability and proteotoxicity. Neuromolecular Med 11:97–100PubMedCrossRefGoogle Scholar
  25. Inoue Y, Kimura A (1995) Methylglyoxal and regulation of its metabolism in microorganisms. Adv Microb Physiol 37:177–227PubMedCrossRefGoogle Scholar
  26. Inoue Y, Maeta K, Nomura W (2011) Glyoxalase system in yeasts: structure, function, and physiology. Semin Cell Dev Biol 22:278–284PubMedCrossRefGoogle Scholar
  27. Kalapos MP (1999) Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications. Toxicol Lett 110:145–175PubMedCrossRefGoogle Scholar
  28. Lanoue FK and Schoolwerth AC (1984) in New Comprehensive Biochemistry, Bioenergetics (Ernster L, ed), pp. 221–268, Elsevier Biomedical Press, AmsterdamGoogle Scholar
  29. Lowry OH, Rosebrough NJ, Farr AL, Randall R (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  30. Maeta K, Mori K, Takatsume Y, Izawa S, Inoue Y (2005) Diagnosis of cell death induced by methylglyoxal, a metabolite derived from glycolysis, in Saccharomyces cerevisiae. FEMS Microbiol Lett 243:87–92PubMedCrossRefGoogle Scholar
  31. Martins AM, Cordeiro C, Freire AP (1999) Glyoxalase II in Saccharomyces cerevisiae: in situ kinetics using the 5,5′-dithiobis(2-nitrobenzoic acid) assay. Arch Biochem Biophys 366:15–20PubMedCrossRefGoogle Scholar
  32. Miyagi H, Kawai S, Murata K (2009) Two sources of mitochondrial NADPH in the yeast Saccharomyces cerevisiae. J Biol Chem 284:7553–7560PubMedCrossRefGoogle Scholar
  33. Moore AL, Bonner WD Jr (1982) Measurements of membrane potentials in plant mitochondria with the safranine. Plant Physiol 70:1271–1276PubMedCrossRefGoogle Scholar
  34. Mourier A, Vallortigara J, Yoboue ED, Rigoulet M, Devin A (2008) Kinetic activation of yeast mitochondrial d-lactate dehydrogenase by carboxylic acids. Biochim Biophys Acta 1777:1283–1288PubMedCrossRefGoogle Scholar
  35. Pallotta ML (2011) Evidence for the presence of a FAD pyrophosphatase and a FMN phosphohydrolase in yeast mitochondria: a possible role in flavin homeostasis. Yeast 28(10):693–705Google Scholar
  36. Pallotta ML, Brizio C, Fratianni A, De Virgilio C, Barile M, Passerella S (1998) Saccharomyces cerevisiae mitochondria can synthesise FMN and FAD from externally added riboflavin and export them to the extramitochondrial phase. FEBS Lett 428:245–249PubMedCrossRefGoogle Scholar
  37. Pallotta ML, Fratianni A, Passarella S (1999) Metabolite transport in isolated yeast mitochondria: fumarate/malate and succinate/malate antiports. FEBS Lett 462:313–316PubMedCrossRefGoogle Scholar
  38. Pallotta ML, Valenti D, Iacovino M, Passarella S (2004) Two separate pathways for d-lactate oxidation by Saccharomyces cerevisiae mitochondria which differ in energy production and carrier involvement. BBA-Bioenergetics 1608:104–113PubMedCrossRefGoogle Scholar
  39. Palmieri L, Runswick MJ, Fiermonte G, Walker JE, Palmieri F (2000) Yeast mitochondrial carriers: bacterial expression, biochemical identification and metabolic significance. J Bioenerg Biomembr 32:67–77PubMedCrossRefGoogle Scholar
  40. Palmieri F, Agrimi G, Blanco E, Castegna A, Di Noia MA, Iacobazzi V, Lasorsa FM, Marobbio CM, Palmieri L, Scarcia P, Todisco S, Vozza A, Walker J (2006) Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins. Biochim Biophys Acta 1757:1249–1262PubMedCrossRefGoogle Scholar
  41. Palmieri F (2008) Diseases caused by defects of mitochondrial carriers: a review. Biochim Biophys Acta 1777:564–578PubMedCrossRefGoogle Scholar
  42. Palmieri F, Pierri CL (2010) Mitochondrial metabolite transport. Essays Biochem 47:37–52PubMedCrossRefGoogle Scholar
  43. Palmieri F, Pierri CL, De Grassi A, Nunes-Nesi A, Fernie AR (2011) Evolution, structure and function of mitochondrial carriers: a review with new insights. Plant J 66:161–181PubMedCrossRefGoogle Scholar
  44. Passarella S, Atlante A, Valenti D, de Bari L (2003) The role of mitochondrial transport in energy metabolism. Mitochondrion 2:319–343PubMedCrossRefGoogle Scholar
  45. Rabbani N, Thornalley PJ (2008) Dicarbonyls linked to damage to the powerhouse: glycation of mitochondrial proteins and oxidative stress. Biochem Soc Trans 38:1045–1050CrossRefGoogle Scholar
  46. Rojo EE, Guiard B, Neupert W, Stuart RA (1998) Sorting of d-lactate dehydrogenase to the inner membrane of mitochondria. Analysis of topogenic signal and energetic requirements. J Biol Chem 273:8040–8047PubMedCrossRefGoogle Scholar
  47. Takatsume Y, Izawa S, Inoue Y (2004) Identification of thermostable glyoxalase I in the fission yeast Schizosaccharomyces pombe. Arch Microbiol 181:371–377PubMedCrossRefGoogle Scholar
  48. Turk Z (2010) Glycotoxins, carbonyl stress and relevance to diabetes and its complications. Physiol Res 59:147–156PubMedGoogle Scholar
  49. Walker ME, Val DL, Rohde M, Devenish RJ, Wallace JC (1991) Yeast pyruvate carboxylase: identification of two genes encoding isoenzymes. Biochem Biophys Res Commun 176:1210–1217PubMedCrossRefGoogle Scholar
  50. Webb BA, Chimenti M, Jacobson MP, Barber DL (2011) Dysregulated pH: a perfect storm for cancer progression. Nat Rev Cancer 11:671–677PubMedCrossRefGoogle Scholar
  51. Wendler A, Irsch T, Rabbani NPJ, Krauth-Siegel RL (2009) Glyoxalase II does not support methylglyoxal detoxification but serves as a general trypanothione thioesterase in African trypanosomes. Mol Biochem Parasitol 163:119–127CrossRefGoogle Scholar
  52. Zara V, Ferramosca A, Capobianco L, Baltz KM, Randel O, Rassow J, Palmieri F, Papatheodorou P (2007) Biogenesis of yeast dicarboxylate carrier: the carrier signature facilitates translocation across the mitochondrial outer membrane. J Cell Sci 120:4099–4106PubMedCrossRefGoogle Scholar
  53. Zhang J, Schneider C, Ottmers L et al (2005) Genomic scale mutant hunt identifies cell size homeostasis genes in S. cerevisiae. Curr Biol 12:1992–2001CrossRefGoogle Scholar
  54. Zinser E, Daum G (1995) Isolation and biochemical characterization of organelles from the yeast, Saccharomyces cerevisiae. Yeast 11:493–536PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.Department of Health SciencesUniversity of MoliseCampobassoItaly

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