Archives of Microbiology

, Volume 195, Issue 1, pp 51–61 | Cite as

The mitochondrial respiratory chain of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill

  • Leobarda Robles-Martínez
  • María Guadalupe Guerra-Sánchez
  • Oscar Flores-Herrera
  • Ana Niurka Hernández-Lauzardo
  • Miguel Gerardo Velázquez-Del Valle
  • Juan Pablo Pardo
Original Paper

Abstract

Rhizopus stolonifer (Ehrenb.:Fr.) Vuill mitochondria contain the complete system for oxidative phosphorylation, formed by the classical components of the electron transport chain (complexes I, II, III, and IV) and the F1F0-ATP synthase (complex V). Using the native gel electrophoresis, we have shown the existence of supramolecular associations of the respiratory complexes. The composition and stoichiometry of the oxidative phosphorylation complexes were similar to those found in other organisms. Additionally, two alternative routes for the oxidation of cytosolic NADH were identified: the alternative NADH dehydrogenase and the glycerol-3-phosphate shuttles. Residual respiratory activity after inhibition of complex IV by cyanide was inhibited by low concentrations of n-octyl gallate, indicating the presence of an alternative oxidase. The K0.5 for the respiratory substrates NADH, succinate, and glycerol-3-phosphate in permeabilized cells was higher than in isolated mitochondria, suggesting that interactions of mitochondria with other cellular elements might be important for the function of this organelle.

Keywords

Oxidative phosphorylation F1F0-ATP synthase Respiratory supercomplexes NADH dehydrogenase Glycerol-3-phosphate shuttle 

Supplementary material

203_2012_845_MOESM1_ESM.jpg (101 kb)
Solubilization of respiratory complexes by DDM. Increased concentrations of DDM were used to release the complexes from Rhizopus stolonifer mitochondria. (A) Blue native gel stained with Coomassie blue and (B) NADH dehydrogenase activity. (JPEG 100 kb)
203_2012_845_MOESM2_ESM.jpg (160 kb)
Solubilization of respiratory supercomplexes by digitonin. Increased concentrations of digitonin were used to release the supercomplexes from Rhizopus stolonifer mitochondria. (A) Blue native gel stained with Coomassie blue and (B) NADH dehydrogenase activity. (JPEG 159 kb)
203_2012_845_MOESM3_ESM.jpg (169 kb)
Respiratory activity and sensitivity by permeabilized Rhizopus stolonifer cells. Cells were permeabilized by digitonin (0.02%) in the absences of any substrate or inhibitor. Oxygen uptake was stimulated by exogenous 384 μM NADH in the (A) absence or (B) presence of 3 μM rotenone, respectively. The respiration was inhibited by 1 mM KCN. Additions are indicated by arrows. Number on the trace represents the rate of oxygen uptake in nmol O2 min-1 (mg cells)-1. Oximetric experiments were performed at 25 °C in buffer A. (JPEG 169 kb)

References

  1. Acin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, Enriquez JA (2008) Respiratory active mitochondrial supercomplexes. Mol Cell 32:529–539PubMedCrossRefGoogle Scholar
  2. Adam SA, Marr RS, Gerace L (1990) Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol 111:807–816PubMedCrossRefGoogle Scholar
  3. Angerer H et al (2011) A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I. Biochem J 437:279–288PubMedCrossRefGoogle Scholar
  4. Averet N, Fitton V, Bunoust O, Rigoulet M, Guerin B (1998) Yeast mitochondrial metabolism: from in vitro to in situ quantitative study. Mol Cell Biochem 184:67–79PubMedCrossRefGoogle Scholar
  5. Averet N, Aguilaniu H, Bunoust O, Gustafsson L, Rigoulet M (2002) NADH is specifically channeled through the mitochondrial porin channel in Saccharomyces cerevisiae. J Bioenerg Biomembr 34:499–506PubMedCrossRefGoogle Scholar
  6. Bakker BM et al (2001) Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae. FEMS Microbiol Rev 25:15–37PubMedCrossRefGoogle Scholar
  7. Bensadoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70:241–250PubMedCrossRefGoogle Scholar
  8. Bianchi C, Genova ML, Parenti Castelli G, Lenaz G (2004) The mitochondrial respiratory chain is partially organized in a supercomplex assembly: kinetic evidence using flux control analysis. J Biol Chem 279:36562–36569PubMedCrossRefGoogle Scholar
  9. Boumans H, Grivell LA, Berden JA (1998) The respiratory chain in yeast behaves as a single functional unit. J Biol Chem 273:4872–4877PubMedCrossRefGoogle Scholar
  10. Cordeiro C, Freire AP (1995a) Digitonin permeabilization of Saccharomyces cerevisiae cells for in situ enzyme assay. Anal Biochem 229:145–148PubMedCrossRefGoogle Scholar
  11. Cordeiro CA, Freire AP (1995b) In situ regulation of methylglyoxal metabolism. Biochem Soc Trans 23:291SPubMedGoogle Scholar
  12. Cruciat CM, Brunner S, Baumann F, Neupert W, Stuart RA (2000) The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria. J Biol Chem 275:18093–18098PubMedCrossRefGoogle Scholar
  13. Elthon TE, Nickels RL, McIntosh L (1989) Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. Plant Physiol 89:1311–1317PubMedCrossRefGoogle Scholar
  14. Eubel H, Heinemeyer J, Braun HP (2004a) Identification and characterization of respirasomes in potato mitochondria. Plant Physiol 134:1450–1459PubMedCrossRefGoogle Scholar
  15. Eubel H, Heinemeyer J, Sunderhaus S, Braun HP (2004b) Respiratory chain supercomplexes in plant mitochondria. Plant Physiol Biochem 42:937–942PubMedCrossRefGoogle Scholar
  16. Fontaine EM, Keriel C, Lantuejoul S, Rigoulet M, Leverve XM, Saks VA (1995) Cytoplasmic cellular structures control permeability of outer mitochondrial membrane for ADP and oxidative phosphorylation in rat liver cells. Biochem Biophys Res Commun 213:138–146PubMedCrossRefGoogle Scholar
  17. Genova ML et al (2008) Is supercomplex organization of the respiratory chain required for optimal electron transfer activity? Biochim Biophys Acta 1777:740–746PubMedCrossRefGoogle Scholar
  18. Guerin B, Labbe P, Somlo M (1979) Preparation of yeast mitochondria (Saccharomyces cerevisiae) with good P/O and respiratory control ratios. Methods Enzymol 55:149–159PubMedCrossRefGoogle Scholar
  19. Guerra-Sánchez MG, Vega-Pérez J, Velázquez-del Valle MG, Hernández-Lauzardo AN (2009) Antifungal activity and release of compounds on Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. by effect of chitosan with different molecular weights. Pestic Biochem Physiol 93:18–22CrossRefGoogle Scholar
  20. Hernández-Lauzardo AN, Bautista-Baños S, Trejo-Espino JL, Velázquez-del Valle MG (2006) Identification of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. causal agent of rhizopus rot disease of fruits and vegetables. Mex J Phytopathol 24:65–69Google Scholar
  21. Joseph-Horne T, Hollomon DW, Wood PM (2001) Fungal respiration: a fusion of standard and alternative components. Biochim Biophys Acta 1504:179–195PubMedCrossRefGoogle Scholar
  22. Juarez O, Guerra G, Martinez F, Pardo JP (2004) The mitochondrial respiratory chain of Ustilago maydis. Biochim Biophys Acta 1658:244–251PubMedCrossRefGoogle Scholar
  23. Juarez O, Guerra G, Velazquez I, Flores-Herrera O, Rivera-Perez RE, Pardo JP (2006) The physiologic role of alternative oxidase in Ustilago maydis. FEBS J 273:4603–4615PubMedCrossRefGoogle Scholar
  24. Jung C, Higgins CM, Xu Z (2000) Measuring the quantity and activity of mitochondrial electron transport chain complexes in tissues of central nervous system using blue native polyacrylamide gel electrophoresis. Anal Biochem 286:214–223PubMedCrossRefGoogle Scholar
  25. Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS (2008) Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc 3:965–976PubMedCrossRefGoogle Scholar
  26. Laouar L, Mulligan BJ, Lowe KC (1992) Yeast permeabilization with surfactants. Biotechnol Lett 14:719–720CrossRefGoogle Scholar
  27. Lenaz G, Genova ML (2009) Structural and functional organization of the mitochondrial respiratory chain: a dynamic super-assembly. Int J Biochem Cell Biol 41:1750–1772PubMedCrossRefGoogle Scholar
  28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  29. McKenzie M, Ryan MT (2010) Assembly factors of human mitochondrial complex I and their defects in disease. IUBMB Life 62:497–502PubMedCrossRefGoogle Scholar
  30. Milner DJ, Mavroidis M, Weisleder N, Capetanaki Y (2000) Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function. J Cell Biol 150:1283–1298PubMedCrossRefGoogle Scholar
  31. Noubhani A, Bunoust O, Rigoulet M, Thevelein JM (2000) Reconstitution of ethanolic fermentation in permeabilized spheroplasts of wild-type and trehalose-6-phosphate synthase mutants of the yeast Saccharomyces cerevisiae. Eur J Biochem 267:4566–4576PubMedCrossRefGoogle Scholar
  32. Paumard P et al (2002) The ATP synthase is involved in generating mitochondrial cristae morphology. EMBO J 21:221–230PubMedCrossRefGoogle Scholar
  33. Ravanel P, Creuzet S, Tissut M (1990) Inhibitory effect of hydroxyflavones on the exogenous nadh dehydrogenase of plant mitochondrial inner membranes. Phytochemistry 29:441–445CrossRefGoogle Scholar
  34. Rigoulet M et al (2004) Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae. Mol Cell Biochem 256–257:73–81PubMedCrossRefGoogle Scholar
  35. Saks VA, Belikova YO, Kuznetsov AV (1991) In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP. Biochim Biophys Acta 1074:302–311PubMedCrossRefGoogle Scholar
  36. Saks VA et al (1993) Retarded diffusion of ADP in cardiomyocytes: possible role of mitochondrial outer membrane and creatine kinase in cellular regulation of oxidative phosphorylation. Biochim Biophys Acta 1144:134–148PubMedCrossRefGoogle Scholar
  37. Saks VA et al (1995) Control of cellular respiration in vivo by mitochondrial outer membrane and by creatine kinase. A new speculative hypothesis: possible involvement of mitochondrial-cytoskeleton interactions. J Mol Cell Cardiol 27:625–645PubMedCrossRefGoogle Scholar
  38. Schagger H (2001) Respiratory chain supercomplexes. IUBMB Life 52:119–128PubMedCrossRefGoogle Scholar
  39. Schagger H, Pfeiffer K (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 19:1777–1783PubMedCrossRefGoogle Scholar
  40. Schagger H, Pfeiffer K (2001) The ratio of oxidative phosphorylation complexes I–V in bovine heart mitochondria and the composition of respiratory chain supercomplexes. J Biol Chem 276:37861–37867PubMedGoogle Scholar
  41. Schagger H, Vonjagow G (1991) Blue native electrophoresis for isolation of membrane-protein complexes in enzymatically active form. Anal Biochem 199:223–231PubMedCrossRefGoogle Scholar
  42. Schipper MA (1984) A revision of the genus Rhizopus. Studies in mycology. Baarn, The NetherlandsGoogle Scholar
  43. Siedow JN, Umbach AL (2000) The mitochondrial cyanide-resistant oxidase: structural conservation amid regulatory diversity. Biochim Biophys Acta 1459:432–439PubMedCrossRefGoogle Scholar
  44. Sierra-Campos E, Velazquez I, Matuz-Mares D, Villavicencio-Queijeiro A, Pardo JP (2009) Functional properties of the Ustilago maydis alternative oxidase under oxidative stress conditions. Mitochondrion 9:96–102PubMedCrossRefGoogle Scholar
  45. Silva AM, Oliveira PJ (2012) Evaluation of respiration with clark type electrode in isolated mitochondria and permeabilized animal cells. Methods Mol Biol 810:7–24PubMedCrossRefGoogle Scholar
  46. Snowdon AL (1992) Color atlas of post-harvest diseases and disorders of fruits and vegetables. CRC Press, Boca RatonGoogle Scholar
  47. Stuart RA (2008) Supercomplex organization of the oxidative phosphorylation enzymes in yeast mitochondria. J Bioenerg Biomembr 40:411–417PubMedCrossRefGoogle Scholar
  48. Tepp K et al (2011) High efficiency of energy flux controls within mitochondrial interactosome in cardiac intracellular energetic units. Biochim Biophys Acta 1807:1549–1561PubMedCrossRefGoogle Scholar
  49. Timmers AC, Reiss HD, Schel JH (1991) Digitonin-aided loading of Fluo-3 into embryogenic plant cells. Cell Calcium 12:515–521PubMedCrossRefGoogle Scholar
  50. Van Etten JL, Bulla LA Jr, St. Julian G (1974) Physiological and morphological correlation of Rhizopus stolonifer spore germination. J Bacteriol 117:882–887PubMedGoogle Scholar
  51. Velazquez I, Pardo JP (2001) Kinetic characterization of the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae. Arch Biochem Biophys 389:7–14PubMedCrossRefGoogle Scholar
  52. Vercesi AE, Bernardes CF, Hoffmann ME, Gadelha FR, Docampo R (1991) Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ. J Biol Chem 266:14431–14434PubMedGoogle Scholar
  53. Vercesi AE, Rodrigues CO, Uyemura SA, Zhong L, Moreno SN (1998) Respiration and oxidative phosphorylation in the apicomplexan parasite Toxoplasma gondii. J Biol Chem 273:31040–31047PubMedCrossRefGoogle Scholar
  54. Videira A, Duarte M (2001) On complex I and other NADH: ubiquinone reductases of Neurospora crassa mitochondria. J Bioenerg Biomembr 33:197–203CrossRefGoogle Scholar
  55. Vonck J, Schafer E (2009) Supramolecular organization of protein complexes in the mitochondrial inner membrane. Biochim Biophys Acta 1793:117–124PubMedCrossRefGoogle Scholar
  56. Wittig I, Beckhaus T, Wumaier Z, Karas M, Schagger H (2010) Mass estimation of native proteins by blue native electrophoresis: principles and practical hints. Mol Cell Proteomics 9:2149–2161PubMedCrossRefGoogle Scholar
  57. Zerbetto E, Vergani L, Dabbeni-Sala F (1997) Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels. Electrophoresis 18:2059–2064PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Leobarda Robles-Martínez
    • 1
  • María Guadalupe Guerra-Sánchez
    • 1
  • Oscar Flores-Herrera
    • 2
  • Ana Niurka Hernández-Lauzardo
    • 3
  • Miguel Gerardo Velázquez-Del Valle
    • 3
  • Juan Pablo Pardo
    • 2
  1. 1.Departamento de Microbiología, Escuela Nacional de Ciencias BiológicasInstituto Politécnico NacionalMexico, D. F.Mexico
  2. 2.Departamento de Bioquímica, Facultad de MedicinaUniversidad Nacional Autónoma de MéxicoMexico, D. F.Mexico
  3. 3.CEPROBI, Instituto Politécnico NacionalYautepecMexico

Personalised recommendations