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

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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.

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References

  • Acin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, Enriquez JA (2008) Respiratory active mitochondrial supercomplexes. Mol Cell 32:529–539

    Article  PubMed  CAS  Google Scholar 

  • Adam SA, Marr RS, Gerace L (1990) Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol 111:807–816

    Article  PubMed  CAS  Google Scholar 

  • 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 437:279–288

    Article  PubMed  CAS  Google Scholar 

  • 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 184:67–79

    Article  PubMed  CAS  Google Scholar 

  • 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 34:499–506

    Article  PubMed  CAS  Google Scholar 

  • Bakker BM et al (2001) Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae. FEMS Microbiol Rev 25:15–37

    Article  PubMed  CAS  Google Scholar 

  • Bensadoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70:241–250

    Article  PubMed  CAS  Google Scholar 

  • 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 279:36562–36569

    Article  PubMed  CAS  Google Scholar 

  • Boumans H, Grivell LA, Berden JA (1998) The respiratory chain in yeast behaves as a single functional unit. J Biol Chem 273:4872–4877

    Article  PubMed  CAS  Google Scholar 

  • Cordeiro C, Freire AP (1995a) Digitonin permeabilization of Saccharomyces cerevisiae cells for in situ enzyme assay. Anal Biochem 229:145–148

    Article  PubMed  CAS  Google Scholar 

  • Cordeiro CA, Freire AP (1995b) In situ regulation of methylglyoxal metabolism. Biochem Soc Trans 23:291S

    PubMed  CAS  Google Scholar 

  • 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 275:18093–18098

    Article  PubMed  CAS  Google Scholar 

  • Elthon TE, Nickels RL, McIntosh L (1989) Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. Plant Physiol 89:1311–1317

    Article  PubMed  CAS  Google Scholar 

  • Eubel H, Heinemeyer J, Braun HP (2004a) Identification and characterization of respirasomes in potato mitochondria. Plant Physiol 134:1450–1459

    Article  PubMed  CAS  Google Scholar 

  • Eubel H, Heinemeyer J, Sunderhaus S, Braun HP (2004b) Respiratory chain supercomplexes in plant mitochondria. Plant Physiol Biochem 42:937–942

    Article  PubMed  CAS  Google Scholar 

  • 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 213:138–146

    Article  PubMed  CAS  Google Scholar 

  • Genova ML et al (2008) Is supercomplex organization of the respiratory chain required for optimal electron transfer activity? Biochim Biophys Acta 1777:740–746

    Article  PubMed  CAS  Google Scholar 

  • Guerin B, Labbe P, Somlo M (1979) Preparation of yeast mitochondria (Saccharomyces cerevisiae) with good P/O and respiratory control ratios. Methods Enzymol 55:149–159

    Article  PubMed  CAS  Google Scholar 

  • 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 93:18–22

    Article  Google Scholar 

  • 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 24:65–69

    Google Scholar 

  • Joseph-Horne T, Hollomon DW, Wood PM (2001) Fungal respiration: a fusion of standard and alternative components. Biochim Biophys Acta 1504:179–195

    Article  PubMed  CAS  Google Scholar 

  • Juarez O, Guerra G, Martinez F, Pardo JP (2004) The mitochondrial respiratory chain of Ustilago maydis. Biochim Biophys Acta 1658:244–251

    Article  PubMed  CAS  Google Scholar 

  • 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 273:4603–4615

    Article  PubMed  CAS  Google Scholar 

  • 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 286:214–223

    Article  PubMed  CAS  Google Scholar 

  • 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 3:965–976

    Article  PubMed  CAS  Google Scholar 

  • Laouar L, Mulligan BJ, Lowe KC (1992) Yeast permeabilization with surfactants. Biotechnol Lett 14:719–720

    Article  CAS  Google Scholar 

  • Lenaz G, Genova ML (2009) Structural and functional organization of the mitochondrial respiratory chain: a dynamic super-assembly. Int J Biochem Cell Biol 41:1750–1772

    Article  PubMed  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  • McKenzie M, Ryan MT (2010) Assembly factors of human mitochondrial complex I and their defects in disease. IUBMB Life 62:497–502

    Article  PubMed  CAS  Google Scholar 

  • Milner DJ, Mavroidis M, Weisleder N, Capetanaki Y (2000) Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function. J Cell Biol 150:1283–1298

    Article  PubMed  CAS  Google Scholar 

  • 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 267:4566–4576

    Article  PubMed  CAS  Google Scholar 

  • Paumard P et al (2002) The ATP synthase is involved in generating mitochondrial cristae morphology. EMBO J 21:221–230

    Article  PubMed  CAS  Google Scholar 

  • Ravanel P, Creuzet S, Tissut M (1990) Inhibitory effect of hydroxyflavones on the exogenous nadh dehydrogenase of plant mitochondrial inner membranes. Phytochemistry 29:441–445

    Article  CAS  Google Scholar 

  • Rigoulet M et al (2004) Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae. Mol Cell Biochem 256–257:73–81

    Article  PubMed  Google Scholar 

  • 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 1074:302–311

    Article  PubMed  CAS  Google Scholar 

  • 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 1144:134–148

    Article  PubMed  CAS  Google Scholar 

  • 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 27:625–645

    Article  PubMed  CAS  Google Scholar 

  • Schagger H (2001) Respiratory chain supercomplexes. IUBMB Life 52:119–128

    Article  PubMed  CAS  Google Scholar 

  • Schagger H, Pfeiffer K (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 19:1777–1783

    Article  PubMed  CAS  Google Scholar 

  • 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 276:37861–37867

    PubMed  CAS  Google Scholar 

  • Schagger H, Vonjagow G (1991) Blue native electrophoresis for isolation of membrane-protein complexes in enzymatically active form. Anal Biochem 199:223–231

    Article  PubMed  CAS  Google Scholar 

  • Schipper MA (1984) A revision of the genus Rhizopus. Studies in mycology. Baarn, The Netherlands

    Google Scholar 

  • Siedow JN, Umbach AL (2000) The mitochondrial cyanide-resistant oxidase: structural conservation amid regulatory diversity. Biochim Biophys Acta 1459:432–439

    Article  PubMed  CAS  Google Scholar 

  • 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 9:96–102

    Article  PubMed  CAS  Google Scholar 

  • Silva AM, Oliveira PJ (2012) Evaluation of respiration with clark type electrode in isolated mitochondria and permeabilized animal cells. Methods Mol Biol 810:7–24

    Article  PubMed  CAS  Google Scholar 

  • Snowdon AL (1992) Color atlas of post-harvest diseases and disorders of fruits and vegetables. CRC Press, Boca Raton

    Google Scholar 

  • Stuart RA (2008) Supercomplex organization of the oxidative phosphorylation enzymes in yeast mitochondria. J Bioenerg Biomembr 40:411–417

    Article  PubMed  CAS  Google Scholar 

  • Tepp K et al (2011) High efficiency of energy flux controls within mitochondrial interactosome in cardiac intracellular energetic units. Biochim Biophys Acta 1807:1549–1561

    Article  PubMed  CAS  Google Scholar 

  • Timmers AC, Reiss HD, Schel JH (1991) Digitonin-aided loading of Fluo-3 into embryogenic plant cells. Cell Calcium 12:515–521

    Article  PubMed  CAS  Google Scholar 

  • Van Etten JL, Bulla LA Jr, St. Julian G (1974) Physiological and morphological correlation of Rhizopus stolonifer spore germination. J Bacteriol 117:882–887

    PubMed  Google Scholar 

  • Velazquez I, Pardo JP (2001) Kinetic characterization of the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae. Arch Biochem Biophys 389:7–14

    Article  PubMed  CAS  Google Scholar 

  • 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 266:14431–14434

    PubMed  CAS  Google Scholar 

  • Vercesi AE, Rodrigues CO, Uyemura SA, Zhong L, Moreno SN (1998) Respiration and oxidative phosphorylation in the apicomplexan parasite Toxoplasma gondii. J Biol Chem 273:31040–31047

    Article  PubMed  CAS  Google Scholar 

  • Videira A, Duarte M (2001) On complex I and other NADH: ubiquinone reductases of Neurospora crassa mitochondria. J Bioenerg Biomembr 33:197–203

    Article  CAS  Google Scholar 

  • Vonck J, Schafer E (2009) Supramolecular organization of protein complexes in the mitochondrial inner membrane. Biochim Biophys Acta 1793:117–124

    Article  PubMed  CAS  Google Scholar 

  • 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 9:2149–2161

    Article  PubMed  CAS  Google Scholar 

  • Zerbetto E, Vergani L, Dabbeni-Sala F (1997) Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels. Electrophoresis 18:2059–2064

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from Consejo Nacional de Ciencia y Tecnología (CONACyT 59855) and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT IN210311-3) from Universidad Nacional Autónoma de México, and Secretaría de Investigación y Posgrado (SIP), Instituto Politécnico Nacional, grants 20070822, 20080183, and 20080561. Leobarda Robles-Martínez is a Ph.D. student in the Quimicobiológicas Program from Escuela Nacional de Ciencias Biológicas of Instituto Politécnico Nacional and was supported by grants from CONACyT (206876) and SIP (Programa Institucional de Formación de Investigadores).

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Authors and Affiliations

  1. Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico, D. F., Mexico

    Leobarda Robles-Martínez & María Guadalupe Guerra-Sánchez

  2. Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apartado Postal 70-159, Coyoacán, 04510, Mexico, D. F., Mexico

    Oscar Flores-Herrera & Juan Pablo Pardo

  3. CEPROBI, Instituto Politécnico Nacional, Yautepec, Morelos, Mexico

    Ana Niurka Hernández-Lauzardo & Miguel Gerardo Velázquez-Del Valle

Authors
  1. Leobarda Robles-Martínez
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  2. María Guadalupe Guerra-Sánchez
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  3. Oscar Flores-Herrera
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  4. Ana Niurka Hernández-Lauzardo
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  5. Miguel Gerardo Velázquez-Del Valle
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  6. Juan Pablo Pardo
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Corresponding author

Correspondence to Juan Pablo Pardo.

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Communicated by John Helman.

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203_2012_845_MOESM1_ESM.jpg

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

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

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)

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Robles-Martínez, L., Guerra-Sánchez, M.G., Flores-Herrera, O. et al. The mitochondrial respiratory chain of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. Arch Microbiol 195, 51–61 (2013). https://doi.org/10.1007/s00203-012-0845-7

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