Abstract
Under anaerobiosis, the mitochondrion of Saccharomyces cerevisiae is restricted to unstructured promitochondria. These promitochondria provide unknown metabolic functions that are required for growth. Since high glucose concentrations are mainly fermented by S. cerevisiae during stationary phase (due to nitrogen starvation), an optimized promitochondria isolation procedure was investigated. Firstly, the unusual promitochondria ultrastructure was checked in intact cells by electron microscopy using a cryo-fixation and freeze-substitution method. The rapid response of anaerobic cells toward oxygen justified the adoption of several critical steps, especially during spheroplasting. Control of spheroplasting was accompanied by a systematic analysis of spheroplast integrity, which greatly influence the final quality of promitochondria. Despite the presence of remnant respiratory chain components under anaerobiosis, characterization of isolated promitochondria by high-resolution respirometry did not reveal any antimycin A- and myxothiazol-sensitive NADH and NADPH oxidase activities. Moreover, the existence of a cyanide-sensitive and non-phosphorylating NADH-dependent oxygen consumption in promitochondria was demonstrated. Nevertheless, promitochondria only slightly contribute to the overall oxygen consumption capacity observed in highly glucose-repressed anaerobic cells.
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Allen L.A., Zhao X.J., Caughey W. and Poyton R.O. 1995 Isoforms of yeast cytochrome c oxidase subunit V affect the binuclear reaction center and alter the kinetics of interaction with the isoforms of yeast cytochrome c. J. Biol. Chem. 270: 110-118.
Andreasen A.A. and Stier T.J.B. 1953. Anaerobic nutrition of Saccharomyces cerevisiae. I. ergosterol requirement for growth in defined medium. J. Cell. Comp. Physiol. 41: 23-36.
Andreasen A.A. and Stier T.J.B. 1954. Anaerobic nutrition of Saccharomyces cerevisiae. II. Unsaturated fatty acid require-ment for growth in defined medium. J. Cell. Comp. Physiol. 43: 271-281.
Aoyama Y., Yoshida Y., Sato R., Susani M. and Ruis H. 1981. Involvement of cytochrome b5 and a cyanide-sensitive mono-oxygenase in the 4-demethylation of 4,4-dimethylzymosterol by yeast microsomes. Biochim. Biophys. Acta 663: 194-202.
Baba M. and Osumi M. 1987 Transmission and scanning electron microscopic examination of intracellular organelles in freeze-substitued Kloeckera and Saccharomyces cerevisiae yeast cells. J. Electron. Microsc. 5: 249-261.
Baker K.P. and Schatz G. 1991. Mitochondrial proteins essential for viability mediate protein transport into yeast mitochondria. Nature 349: 205-208.
Baumgartner U., Hamilton B., Piskacek M., Ruis H. and Rottensteiner H. 1999. Functional analysis of the Zn(2)Cys(6) transcription factors Oaf1p and Pip2p. Different roles in fatty acid induction of beta-oxidation in Saccharomyces cerevisiae. J. Biol. Chem. 274: 22208-22216.
Beauvoit B., Bunoust O., Guerin B. and Rigoulet M. 1999. ATP-root regulation of cytochrome oxidase in yeast mitochondria. Role of subunit VIa. Eur. J. Biochem. 263: 118-127.
Bely M., Sablayrolles J.M. and Barre P. 1990. Description of alcoholic fermentation kinetics: its variability and significance. Am. J. Enol. Vitic. 40: 319-324.
Bertrand J.C., Mattei G., Parra C., Giordani R. and Gilewicz M. 1984. Influence of oxygen on the microsomal electron transport system in Saccharomyces cerevisiae. Biochimie 66: 583-538.
Boles E., de Jong-Gubbels P. and Pronk J.T. 1998. Identification and characterization of MAE1, the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme. J. Bacteriol. 180: 2875-2882.
Bruinenberg P.M., van Dijken J.P., Kuenen J.G. and Scheffers W.A. 1985. Critical parameters in the isolation of mitochondria from Candida utilis grown in continuous culture. J. Gen. Microbiol. 131: 1035-1042.
Burke P.V., Raitt D.C., Allen L.A., Kellog E.A. and Poyton R.O. 1997. Effect of oxygen concentration on the expression of cytochrome c and cytochrome c oxidase genes in yeast. J. Biol. Chem. 272: 14705-14712.
Cartledge T.G. and Lloyd D. 1972. Subcellular fractionation by zonal centrifugation of glucose repressed anaerobically grown Saccharomyces carlbergensis. Biochem. J. 127: 693-703.
Criddle R.S. and Schatz G. 1969. Promitochondria of anaerobically cytogrown yeast. I. Isolation and biochemical properties. Biochemistry 8: 322-334.
Dagsgaard C., Taylor L.E., O'Brien K.M. and Poyton R.O. 2001. Effects of anoxia and the mitochondrion on expression of aerobic nuclear COX genes in yeast: evidence for a signaling pathway from the mitochondrial genome to the nucleus. J. Biol. Chem. 276: 7593-7601.
Damsky C.H., Nelson W.M. and Claude A. 1969. Mitochondria in mitochonanaerobically grown, lipid-limited brewer's yeast. J. Cell. Biol. 43: 174-186.
Damsky C.H. 1976. Environmentally induced changes in mitochondria and endoplasmic reticulum of Saccharomyces Carlsbergensis yeast. J. Cell. Biol. 71: 123-135.
Daum G., Bohni P.C. and Schatz G. 1982. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. Biol. Chem. 257: 13028-1303.
Daum G., Lees N.D., Bard M. and Dickson R. 1998. Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 14: 1471-1510.
De Nobel J.G. and Barnett J.A. 1991. Passage of molecules through yeast cell walls: a brief essay-review. Yeast 7: 313-323.
Dupont C.H., Mazat J.P. and Guerin B. 1985. The role of adenine nucleotide translocation in the energization of the inner membrane of mitochondria isolated from rho and rho strains Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 132: 1116-1123.
Fleet G.H. 1991 Cell walls. In: Rose A.H. and Harrison J.S. (Eds) The Yeasts, Vol 4, 2nd edn, Academic Press, London. pp. 199-277.
Gaigg B., Simbeni R., Hrastnik C., Paltauf F. and Daum G. 1995. Characterization of a microsomal sub-fraction associated with mitochondria of the yeast Saccharomyces cerevisiae. Involvement in synthesis and import of phospholipids into mitochondria. Biochim. Biophys. Acta 1234: 214-220.
Groot G.S., Kovac L. and Schatz G. 1971. Promitochondria anaerobically grown yeast.V. Energy transfer in the absence an electron transfer chain. Proc. Natl. Acad. Sci. U.S.A. 68: 308-311.
Guérin B., Labbe P. and Somlo M. 1979. Preparation of yeast mitochondria (Saccharomyces cerevisiae) with good P/O and respiratory control ratios. Methods Enzymol. 55: 149-159.
Haller T., Ortner M. and Gnaiger E. 1994. A respirometer for investigating oxidative cell metabolism: towards optimization respiratory studies. Anal. Biochem. 218: 338-342.
Heilmann H.D. and Lingens F. 1968. On the regulation of nicotinic acid biosynthesis in Saccharomyces cerevisiae. Hoppe Seylers Z. Physiol. Chem. 349: 231-236.
Ishidate K., Kawaguchi K. and Tagawa K. 1969. Change in P-450 content accompanying aerobic formation of mitochondria yeast. J. Biochem. 65: 385-392.
Jauniaux J.C., Urrestarazu L.A. and Wiame J.M. 1978. Arginine metabolism in Saccharomyces cerevisiae: subcellular localization of the enzymes. J. Bacteriol. 133: 1096-1107.
Kawaguchi K., Ishidate K. and Tagawa K. 1973. Non-mitochondrial electron transfer system in anaerobically grown yeast cells. J. Biochem. (Tokyo) 74: 817-826.
Kubota S., Yoshida Y. and Kumaoka H. 1977. Studies on the microsomal electron-transport system of anaerobically grown yeast. IV. Purification and characterization of NADH-cytogrown chrome b5 reductase. J. Biochem. 81: 187-195.
Kwast K.E., Burke P.V. and Poyton R.O. 1998. Oxygen sensing and the transcriptional regulation of oxygen-responsive genes yeast. J. Exp. Biol. 20: 1177-1195.
Lawson J.E., Gawaz M., Klingenberg M. and Douglas M.G. 1990. Structure-function studies of adenine nucleotide transport mitochondria. I. Construction and genetic analysis of yeast mutants encoding the ADP/ATP carrier protein of mitochondria. J Biol. Chem. 265: 14195-14201.
Liu Z. and Butow R.A. 1999. A transcriptional switch in the expression of yeast tricarboxylic acid cycle genes in response a reduction or loss of respiratory function. Mol. Cell. Biol. 19: 6720-6728.
Lowdon M.J., Gordon P.A. and Stewart P.R. 1972. Regulation the synthesis of mitochondrial enzymes and cytochromes. Distinction between catabolite repression and anaerobiosis in Saccharomyces cerevisiae. Arch. Mikrobiol. 85: 355-361.
Lowry R.H., Rosenbrough N.J., Farr A.L. and Randall R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275.
Maaheimo H., Fiaux J., Cakar Z.P., Bailey J.E., Sauer U. and Szyperski T. 2001. Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional 13C labeling of common amino acids. Eur. J. Biochem. 268: 2464-2479.
Marchant R. and Smith D.G. 1968. Membranous structures in yeast. Biol. Rev. 43: 459-471.
Miller G.L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chem. 31: 426-428.
Nissen T.L., Schulze U., Nielsen J. and Villadsen J. 1997. Flux distributions in anaerobic, glucose-limited continuous cultures of Saccharomyces cerevisiae. Microbiology 143: 203-218.
Osumi T., Nishino T. and Katsuki H. 1979. Studies on the delta 5-desaturation in ergosterol biosynthesis in yeast. J. Biochem. 85: 819-826.
Paltauf F. and Schatz G. 1969. Promitochondria of anaerobically grown yeast II. Lipid composition. Biochemistry 8: 335-339.
Plattner H. and Schatz G. 1969. Promitochondria of anaerobically grown yeast. 3. Morphology. Biochemistry 8: 339-343.
Plattner H., Salpeter M., Saltzgaber J. and Schatz G. 1970. Promitochondria of anaerobically grown yeast IV. Conversion into respiring mitochondria. Proc. Natl. Acad. Sci. USA 66: 1252-1271.
Plattner H., Salpeter M., Saltzgaber J., Rouslin W. and Schatz G. 1971 Pro-mitochondria of anaerobically-grown yeast: evidence Scheffor their conversion into functional mitochondria during respiratory adaptation. In: Boardman N.K., Linnane A.W. and Smillie R.M. (Eds) Autonomy and Biogenesis of Mitochondria and Chloroplasts. North-Holland publishing Company, Amsterdam. pp. 175-184.
Rogers P.J. and Stewart P.R. 1973. Respiratory development in Saccharomyces cerevisiae grown at controlled oxygen tension. J. Bacteriol. 115: 88-97.
Rosenfeld E., Beauvoit B., Rigoulet M. and Salmon J.M. 2002. Non-respiratory oxygen consumption pathways in anaerobically-grown Saccharomyces cerevisiae: evidence and partial characterization. Yeast 19: 1299-1322.
Rosenfeld E., Beauvoit B., Blondin B. and Salmon J.M. 2003. Oxygen consumption by anaerobic Saccharomyces cerevisiae in mitoenological conditions: effect on fermentation kinetics. Appl. Env. Microbiol. 69: 113-121.
Sablayrolles J.M., Dubois C., Manginot C., Roustan J.L. and Barre P. 1996. Effectiveness of combined ammoniacal nitrogen and oxygen additions for completion of sluggish and stuck wine fermentations. J. Ferm. Bioeng. 82: 377-381.
Salmon J.M., Fornairon C. and Barre P. 1998. Determination of oxygen utilization pathways in an industrial strain of Saccharomyces cerevisiae during enological fermentation. J. Ferment. Bioeng. 86: 154-163.
Schatz G. 1965. Subcellular particles carrying mitochondrial en-zymes in anaerobically-grown cells of Saccharomyces cerevisiae. Biochim. Biophys. Acta 96: 342-345.
Shimoi H., Kitagaki H., Ohmori H., Iimura Y. and Ito K. 1998. Sed1p is a major cell wall protein of Saccharomyces cerevisiae in the stationary phase and is involved in lytic enzyme resistance. J. Bacteriol. 180: 3381-3387
Skoneczny M. and Rytka J. 1996. Maintenance of the peroxisomal compartment in glucose-repressed and anaerobically grown Saccharomyces cerevisiae cells. Biochimie. 78: 95-102.
Slonimski P.P., Perrodin G. and Croft J.H. 1968. Ethidium bromide-induced mutation of yeast mitochondria: complete transformation of cells into respiratory deficient nonchromosomal « petites ». Biochem. Biophys. Res. Commun. 30: 232-2391.
Somlo M. 1968. Induction and repression of mitochondrial ATPase in yeast. Eur. J. Biochem. 5: 276-284.
Srere P.A. 1969. Citrate synthase. Methods Enzymol. 13: 3-11.
Stuart R.A., Gruhler A., van der Klei I., Guiard B., Koll H. and Neupert W. 1994. The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and inter-membrane space. Eur. J. Biochem. 220: 9-18.
Subík J., Kolarov J. and Ková? L.K. 1972. Obligatory requirement of intramitochondrial ATP for normal functioning of the eukaryotic cell. Biochem. Biophys. Res. Commun. 49: 192-198.
Trocha P.J. and Sprinson D.B. 1976. Location and regulation of early enzymes of sterol biosynthesis in yeast. Arch. Biochem. Biophys. 174: 45-51.
Visser W., van der Baan A.A., Batenburg-van derVegte W., Scheffers W., Kramer R. and van Dijken J.P. 1994. Involvement of mitochondria in the assimilatory metabolism of anaerobic Saccharomyces cerevisiae cultures. Microbiology 140: 3039-3046.
Visser W., von Spronsen E.A., Nanninga N., Pronk J.T., Gijs Kuenen J. and van Dijken J.P. 1995. Effects of growth conditions on mitochondrial morphology in Saccharomyces cerevisiae. Antonie van Leeuwenhoek 67: 243-253.
Wales D.S., Cartledge T.G. and Lloyd D. 1980. Effects of glucose repression and anaerobiosis on the activities and subcellular distribution of tricarboxylic acid cycle and associated enzymes in Saccharomyces carlsbergensis. J. Gen. Microbiol. 116: 93-98.
Williamson D.H. and Fennell D.J. 1975. The use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA. Meth. Cell. Biol. 12: 335-351.
Yoshida Y., Kumaoka H. and Sato R. 1974. Studies on the microsomal electron-transport system of anaerobically grown yeast. I. Intracellular localization and characterization. J. Biochem. 75: 1201-1210.
Zinser E. and Daum G. 1995. Isolation and biochemical characterization of organelles from the yeast Saccharomyces cerevisiae. Yeast 11: 493-536.
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Rosenfeld, E., Schaeffer, J., Beauvoit, B. et al. Isolation and properties of promitochondria from anaerobic stationary-phase yeast cells. Antonie Van Leeuwenhoek 85, 9–21 (2004). https://doi.org/10.1023/B:ANTO.0000020268.55350.54
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DOI: https://doi.org/10.1023/B:ANTO.0000020268.55350.54