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Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae
FEMS Microbiology Letters 215 (2002) 103^108
www.fems-microbiology.org
Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae Keiichiro Enomoto a , Yukihiko Arikawa b , Haruhiro Muratsubaki a
a;
Department of Clinical Biochemistry, Kyorin University School of Health Sciences, 476 Miyashita, Hachioji, Tokyo 192-8508, Japan b Food Technology Research Institute of Nagano Prefecture, 205-1 Nishibanba, Kurita, Nagano City 380-0921, Japan Received 3 July 2002; received in revised form 7 August 2002 ; accepted 9 August 2002 First published online 12 September 2002
Abstract In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NADþ . When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions. 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Fumarate reductase ; Saccharomyces cerevisiae ; Redox balancing ; Methylene blue ; Mitochondrion
1. Introduction Soluble fumarate reductase from yeast irreversibly catalyzes the conversion of fumarate to succinate and requires FADH2 , FMNH2 , reduced ribo£avin, or reduced forms of some leuco-pigments as electron donors [1^3]. Fumarate reductase is a soluble enzyme containing one molecule of non-covalently bound FAD per molecule of protein. The yeast Saccharomyces cerevisiae is known to contain two isoenzymes of fumarate reductase [4]. One of them is FRDS1, located in the cytosol and encoded by the FRDS gene on chromosome V [4,5]. The other is FRDS2, located in the promitochondria and encoded by the OSM1 gene on chromosome X [4]. The properties of these isoenzymes are quite distinct from those found in Escherichia coli [6,7] and Vibrio succinogenes [8], which are membrane-bound enzymes having a low level of succinate oxidation activity and covalently bound FAD.
* Corresponding author. Tel. : +81 (426) 91-0011; Fax : +81 (426) 91-1094. E-mail address : [email protected] (H. Muratsubaki).
Recently, we constructed disruptants of the FRDS and OSM1 genes [9,10]. The OSM1 disruptant (DOSM) grew more slowly than DBY747 under anaerobic conditions, and the disruptant of both the FRDS and OSM1 genes (DFRDOSM) failed to grow at all. On the other hand, these disruptants have the ability to grow under aerobic conditions. These facts indicate that fumarate reductase isoenzymes are essential for anaerobiosis. During anaerobic growth of S. cerevisiae, energy is derived exclusively from substrate-level phosphorylation in glycolysis. In such cells, the reoxidation of NADH formed during glycolysis does not occur through the respiratory chain that transfers the reducing equivalents to oxygen. Therefore, maintaining a redox balance is important for continuous operation of the catabolic reaction during anaerobiosis. The soluble fumarate reductase from yeast requires FADH2 , FMNH2 or reduced ribo£avin as an electron donor. The reduced equivalents of the reduced £avin nucleotides are probably supplied by NADH. In the present paper, we examined the e¡ect of arti¢cial electron acceptors, which oxidize non-enzymatically cellular NADH to NADþ , on anaerobic growth of cells containing deletion mutations in fumarate reductase. We found that the addition of oxidized
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forms of methylene blue or phenazine methosulfate overcame the inhibition of anaerobic growth of the disruptant of both the FRDS and OSM1 genes. From the above results we concluded that both fumarate reductases participate in the reoxidation of NADH generated by anaerobic metabolism.
2. Materials and methods 2.1. Strains The S. cerevisiae strains used in this study are listed in Table 1. 2.2. Media and growth conditions The disruptants of fumarate reductase genes were cultured in a chemically de¢ned medium, which contained 0.125% yeast nitrogen base (without amino acids and ammonium sulfate) (Difco), 5% glucose, 1.25% ammonium sulfate, and appropriate amounts of required amino acids. Strain DSDHH was grown in YPD medium containing 2% bactopeptone, 1% yeast extract, and 4% glucose. For anaerobic cultivation, ergosterol (0.025 g l31 ) and Tween 80 (1.05 g l31 ) were added to the medium. Other conditions of cultivation were as described previously [2]. To prepare agar plates, 1.5% Difco bacto agar was added to the de¢ned medium. The agar plates were incubated at 30‡C using AnaerobicAnaero Pack for Cell (Mitsubishi Gas Chemical, Japan) in an anaerobic chamber. 2.3. Assays of cell growth and oxidized methylene blue The concentration of oxidized methylene blue in the supernatant obtained after centrifugation of culture and cell growth was determined by measuring absorbance at 600 nm and 660 nm, respectively, using a spectrophotometer V-520 (Jasco, Japan). 2.4. Determination of succinate in culture broth The cultures collected during the exponential growth phase and stationary phase were centrifuged at 4000Ug for 20 min at 4‡C. The resulting supernatant was used to
determine succinate levels. Succinate was assayed using an HPLC organic acid analysis system (Jasco, Japan). 2.5. Fractionation of mitochondria Strain DSDHH was grown aerobically as described above. Mitochondria were prepared and fractionated as described previously [11,12]. The purity of the obtained fraction was judged from the activity of marker enzymes. The enzymatic activity was assayed for myokinase [13], citrate synthase [14], malate dehydrogenase [15], cytochrome c oxidase [16], and kynurenine hydroxylase [17]. Fumarate reductase activity was assayed as described previously [2]. Protein was assayed according to the method of Bradford [18] using a commercial kit (Pierce) with bovine albumin as a standard. 2.6. Chemicals Methylene blue was purchased from Kanto Chemical Co., Inc ; phenazine methosulfate from Sigma ; and luciferase and NAD(P)H:FMN oxidoreductase from Roche Diagnostics GmbH. All other chemicals were of reagent grade.
3. Results and discussion Under anaerobic conditions, strain DFRDOSM, which was simultaneously disrupted for both the FRDS and OSM1 genes, did not grow in the chemically de¢ned agar medium (Fig. 1B). Under such conditions, strain DOSM deleted for the OSM1 gene grew poorly, while strain DFRDS deleted for the FRDS gene as well as the wild-type strain DBY747 grew well (B). These results are consistent with those of previous studies using a YPD medium consisting of rich nutrients [9]. The addition of 2 mM methylene blue (C) or 5 mM phenazine methosulfate (D) to the agar medium fully restored anaerobic growth of strain DFRDOSM. Under aerobic cultivation, strain DBY747 and all the disruptants of fumarate reductase genes grew in the agar medium without any electron acceptors (A) or with 2 mM methylene blue or 5 mM phenazine methosulfate (data not shown). Strain DFRDOSM was anaerobically cultured in the
Table 1 S. cerevisiae strains used in this study Strain
Genotype
DBY747 DFRDS DOSM DFRDOSM DSDHH
K, K, K, K, K,
a
Source
his3-v1,leu2-3,112,trp1-289a,ura3-52 his3-v1,leu2-3,112,trp1-289a,FRDS : :URA3 his3-v1,leu2-3,112,trp1-289a,OSM1: :URA3 his3-v1,leu2-3,112,trp1-289a,FRDS : :URA3, OSM1: :THAr SDH1: :HIS3,leu2-3,112,trp-289a,ura3-52
The Yeast Genetic Stock Center.
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YGSCa [9] [9] [9] [10]
K. Enomoto et al. / FEMS Microbiology Letters 215 (2002) 103^108
chemically de¢ned liquid medium containing various concentrations of methylene blue. In the presence of 0.01 mM and 0.04 mM methylene blue, DFRDOSM grew exponentially until the oxidized methylene blue was completely exhausted (40 h); thereafter, the cell density was maintained at constant levels until 70 h (Fig. 2). The maximum cell density depended on the concentration of methylene blue added. However, the cell density was not proportional to the concentration of methylene blue. It is known that the arti¢cial electron acceptor methylene blue, oxidizes cellular NADH to NADþ [19]. Phenazine methosulfate also oxidizes NADH to NADþ in vitro and is used as a redox indicator for detection of the dehydrogenase reaction. Accordingly, fumarate reductase isoenzymes FRDS1 and FRDS2 allow for reoxidation of cellular NADH generated by anaerobic metabolism.
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Table 2 Production of succinate by disruptants during anaerobiosis
DBY747 DFRDS DOSM DFRDOSM
Without methylene blue (mmol l31 )
0.08 mM methylene blue (mmol l31 )
0.61 0.60 0.60 ^
0.48 0.51 0.63 0.07
Strains DBY747, DFRDS, DOSM, and DFRDOSM were anaerobically cultured in chemically de¢ned liquid medium with or without 0.08 mM methylene blue. The culture was withdrawn at 70 h in the stationary phase. In all the cultivations, the oxidized methylene blue was completely exhausted at 70 h. The concentration of succinate in the supernatant was determined and expressed as mmol l31 .
Fig. 1. The e¡ect of arti¢cial electron acceptors on growth of disruptants under anaerobic conditions. Strains DBY747 (1), DFRDOSM (2), DOSM (3), and DFRDS (4) were aerobically cultured at 30‡C for 5 days in chemically de¢ned agar medium (A). Under anaerobic conditions, the strains were cultured without electron acceptors (B) or with 2 mM methylene blue (C) or 5 mM phenazine methosulfate (D).
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Fig. 2. The e¡ects of various concentrations of oxidized methylene blue on anaerobic growth of strain DFRDOSM. Strain DFRDOSM was cultured at 30‡C in chemically de¢ned liquid medium with 0 mM (E), 0.01 mM (O), 0.04 mM (U) and 0.08 mM (a) of methylene blue. Cell density (A) during cultivation was determined by measuring absorbance at 660 nm. The concentration of oxidized methylene blue (B) was determined by measuring absorbance at 600 nm.
The production of succinate by fumarate reductase disruptants during anaerobic growth in the presence or absence of methylene blue was studied. In the stationary phase, succinate production by strain DFRDOSM was markedly decreased, corresponding to 21% of that of strain DBY747 (Table 2). However, the production in strains DFRDS and DOSM was unchanged from that in the DBY747 strain. These results indicate that, under anaerobic conditions, succinate is produced by the joint activity of the two fumarate reductase isoenzymes, FRDS1 and FRDS2. The metabolism of S. cerevisiae as well as other eukaryotic cells cannot be understood without considering metabolic compartmentalization. FRDS2 and FRDS1 are known to exist in promitochondria and in the cytosol, respectively [4]. We further studied the detailed distribution of FRDS2 in mitochondria obtained from aerobically grown cells. We did not use anaerobically grown cells possessing mitochondria-like particles, which have been
designated promitochondria [20]. The reason for this is that promitochondria lack respiratory enzymes used as markers of fractionation [20]. To avoid interference by mitochondrial succinate dehydrogenase, which catalyzes not only the succinate oxidation but also the fumarate reduction, strain DSDHH deleted for the SDH1 gene encoding the £avoprotein subunit of succinate dehydrogenase [21] was utilized for determining the localization of FRDS2. Mitochondria were fractionated into three fractions: membrane (inner and outer membranes), intermembrane space, and matrix. The activities of fumarate reductase and marker enzymes in each fraction were determined. As shown in Fig. 3, a large portion of fumarate reductase activity was located in the matrix of the mitochondria. Since mitochondria are not permeable to pyridine nucleotides, including NADH, some device must exist to allow for the reoxidation of the reduced nucleotides in the compartment where they are generated. In aerobically
Fig. 3. Intramitochondrial localization of FRDS2. Strain DSDHH was grown aerobically in YPD medium. Marker enzymes used to judge the purity of the obtained fraction were kynurenine hydroxylase (KHY), cytochrome c oxidase (COX), myokinase (MKI), citrate synthase (CSY), and malate dehydrogenase (MDH). The abscissa denotes the percentages of total activity in mitochondria.
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grown cells, the excess NADH produced in the assimilation process can be reoxidized by mitochondrial respiration. However, under anaerobic conditions, respiration is inoperative. In anaerobic growth, a method for continuously regenerating the NADþ needed for glycolysis is required, because the cellular pool of pyridine nucleotide coenzymes is limited. In the glycolytic pathway, the reduction of NADþ in the glyceraldehyde-3-phosphate dehydrogenase reaction is stoichiometrically linked to the oxidation of NADH in the alcohol dehydrogenase reaction. Therefore, ethanol formation cannot account for the reoxidation of assimilatory NADH. Several reaction processes can perform this role of regenerating NADþ . As described previously, promitochondria lack an integrated electron transfer chain, a functional oxidative phosphorylation system, and several respiratory enzymes [20]. In such cells, therefore, the citrate cycle does not appear to proceed in a cyclic manner. Nissen et al. [22] showed that a large part of the mitochondrial NADH is produced from the synthesis of 2-oxoglutarate, which is a precursor of glutamate. In the present study, we found that the inhibition of the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was overcome by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NADþ . We conclude that in anaerobically grown cells, FRDS2 in the promitochondria matrix and FRDS1 in the cytosol are involved in the reoxidation of excess NADH in each compartment. Soluble fumarate reductase from yeast requires FADH2 and FMNH2 , but not NADH, as electron donors for fumarate reduction. Thus, the enzyme catalyzing the conversion of the oxidized £avin nucleotides by NADH to the reduced forms is required for the reoxidation of NADH by fumarate reductase. At the present time, the enzyme in S. cerevisiae possessing such characteristics remains to be identi¢ed. Other ways to maintain the redox balance during anaerobiosis are known in S. cerevisiae. Cytosolic GPD2, which is one of the glycerol-3-phosphate dehydrogenase isoenzymes, is important for the reoxidation of the cytosolic NADH that migrates from mitochondria [23]. Under anaerobic conditions, a GPD2-deleted mutant grows more slowly and accumulates less glycerol than the wild-type strain. Also, mitochondrial ADH3, one of the alcohol dehydrogenases, may play a key role in the reoxidation of NADH in anaerobically grown cells [24,25]. The anaerobic growth of the ADH3-deletion mutant was slower than that of the wild strain. The ethanol produced in mitochondria by ADH3 would be expected to di¡use into the cytosol, resulting in the transport of excess intramitochondrial NADH to the cytosol [26]. Fumarate reductase, glycerol3-phosphate dehydrogenase, and alcohol dehydrogenase may cooperatively function in maintaining the redox balance during anaerobiosis.
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References [1] Rossi, C., Hauber, J. and Singer, T.P. (1964) Mitochondrial and cytoplasmic enzymes for the reduction of fumarate to succinate in yeast. Nature 204, 167^170. [2] Muratsubaki, H. and Katsume, T. (1982) Puri¢cation and properties of fumarate reductase from baker’s yeast. Agric. Biol. Chem. 46, 2909^2917. [3] Muratsubaki, H. and Katsume, T. (1985) Characterization of fumarate reductase from baker’s yeast: Essential sulfhydryl group for binding of FAD. J. Biochem. 97, 1201^1209. [4] Muratsubaki, H. and Enomoto, K. (1998) One of the fumarate reductase isoenzymes from Saccharomyces cerevisiae is encoded by the OSM1 gene. Arch. Biochem. Biophys. 352, 175^181. [5] Enomoto, K., Ohki, R. and Muratsubaki, H. (1996) Cloning and sequencing of the gene encoding the soluble fumarate reductase from Saccharomyces cerevisiae. DNA Res. 3, 263^267. [6] Weiner, J.H. and Dickie, P.J. (1979) Fumarate reductase of Escherichia coli. Elucidation of the covalent-£avin component. J. Biol. Chem. 254, 8590^8593. [7] Van Hellemond, J.J. and Tielens, A.G.M. (1994) Expression and functional properties of fumarate reductase. Biochem. J. 304, 321^ 331. [8] Unden, G., Hackenberg, H. and Kroger, A. (1980) Isolation and functional aspects of the fumarate reductase involved in the phosphorylative electron transport of Vibrio succinogenes. Biochim. Biophys. Acta 591, 275^288. [9] Arikawa, Y., Enomoto, K., Muratsubaki, H. and Okazaki, M. (1998) Soluble fumarate reductase isoenzymes from Saccharomyces cerevisiae are required for anaerobic growth. FEMS Microbiol. Lett. 165, 111^116. [10] Arikawa, Y., Kuroyanagi, T., Shimosaka, M., Muratsubaki, H., Enomoto, K., Kodaira, R. and Okazaki, M. (1999) E¡ect of gene disruptions of the TCA cycle on production of succinic acid in Saccharomyces cerevisiae. J. Biosci. Bioeng. 87, 28^36. [11] De Kroon, A.I.P.M., Koorengevel, M.C., Goerdayal, S.S., Mulders, P.C., Janssen, M.J.F.W. and De Kruij¡, B. (1999) Isolation and characterization of highly puri¢ed mitochondrial outer membranes of the yeast Saccharomyces cerevisiae (Methods). Mol. Membr. Biol. 16, 205^211. [12] Daum, G., Bo«hni, P.C. and Schatz, G. (1982) Import of proteins into mitochondria. J. Biol. Chem. 257, 13028^13033. [13] Tsuboi, K.K. (1978) AMP (dAMP) kinase from human erythrocytes. Methods Enzymol. 51, 467^473. [14] Parvin, R. (1969) Citrate synthase from yeast. Methods Enzymol. 13, 16^19. [15] Yosida, A. (1969) L-malate dehydrogenase from Bacillus subtilis. Methods Enzymol. 13, 141^144. [16] Mason, T.L., Poyton, R.O., Wharton, D.C. and Schatz, G. (1973) Cytochrome c oxidase from bakers’ yeast. J. Biol. Chem. 248, 1346^ 1354. [17] Bandlow, W. (1972) Membrane separation and biogenesis of the outer membrane of yeast mitochondria. Biochim. Biophys. Acta 282, 105^122. [18] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [19] Khan, S. and O’Brien, P.J. (1995) Modulating hypoxia-induced hepatocyte injury by a¡ecting intracellular redox state. Biochim. Biophys. Acta 1269, 153^161. [20] Criddle, R.S. and Schatz, G. (1969) Promitochondria of anaerobically grown yeast. I. Isolation and biochemical properties. Biochemistry 8, 322^334. [21] Chapman, K.B., Solomon, S.D. and Boeke, J.D. (1992) SDH1, the gene encoding the succinate dehydrogenase £avoprotein subunits from Saccharomyces cerevisiae. Gene 118, 131^136.
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[22] Nissen, T.L., Schulze, J.N. and Villadsen, J. (1997) Flux distribution in anaerobic glucose-limited continuous cultures of Saccharomyces cerevisiae. Microbiology 143, 203^218. [23] Ansell, R., Granath, K., Hohmann, S., Thevelein, J.M. and Adler, L. (1997) The two isoenzymes for yeast NADþ -dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation. EMBO J. 16, 2179^2187. [24] Bakker, B.M., Overkamp, K.M., Van Maris, A.J.A., Ko«tter, P., Luttik, M.A.H., Van Dijiken, J.P. and Pronk, J.T. (2001) Stoichiometry
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and compartmentation of NADH metabolism in Saccharomyces cerevisiae. FEMS Microbiol. Rev. 25, 15^37. [25] Larsson, C., Pafihlman, I.L., Ansell, R., Rigoulet, M., Adler, L. and Gustafsson, L. (1998) The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae. Yeast 14, 347^357. [26] Bakker, B.M., Bro, C., Ko«ttre, P., Luttik, M.A.H., Van Dijken, J.P. and Pronk, J.T. (2000) The mitochondrial alcohol dehydrogenase Adh3p is involved in a redox shuttle in Saccharomyces cerevisiae. J. Bacteriol. 182, 4730^4737.
Cyaan Magenta Geel Zwart
www.fems-microbiology.org
Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae Keiichiro Enomoto a , Yukihiko Arikawa b , Haruhiro Muratsubaki a
a;
Department of Clinical Biochemistry, Kyorin University School of Health Sciences, 476 Miyashita, Hachioji, Tokyo 192-8508, Japan b Food Technology Research Institute of Nagano Prefecture, 205-1 Nishibanba, Kurita, Nagano City 380-0921, Japan Received 3 July 2002; received in revised form 7 August 2002 ; accepted 9 August 2002 First published online 12 September 2002
Abstract In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NADþ . When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions. 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Fumarate reductase ; Saccharomyces cerevisiae ; Redox balancing ; Methylene blue ; Mitochondrion
1. Introduction Soluble fumarate reductase from yeast irreversibly catalyzes the conversion of fumarate to succinate and requires FADH2 , FMNH2 , reduced ribo£avin, or reduced forms of some leuco-pigments as electron donors [1^3]. Fumarate reductase is a soluble enzyme containing one molecule of non-covalently bound FAD per molecule of protein. The yeast Saccharomyces cerevisiae is known to contain two isoenzymes of fumarate reductase [4]. One of them is FRDS1, located in the cytosol and encoded by the FRDS gene on chromosome V [4,5]. The other is FRDS2, located in the promitochondria and encoded by the OSM1 gene on chromosome X [4]. The properties of these isoenzymes are quite distinct from those found in Escherichia coli [6,7] and Vibrio succinogenes [8], which are membrane-bound enzymes having a low level of succinate oxidation activity and covalently bound FAD.
* Corresponding author. Tel. : +81 (426) 91-0011; Fax : +81 (426) 91-1094. E-mail address : [email protected] (H. Muratsubaki).
Recently, we constructed disruptants of the FRDS and OSM1 genes [9,10]. The OSM1 disruptant (DOSM) grew more slowly than DBY747 under anaerobic conditions, and the disruptant of both the FRDS and OSM1 genes (DFRDOSM) failed to grow at all. On the other hand, these disruptants have the ability to grow under aerobic conditions. These facts indicate that fumarate reductase isoenzymes are essential for anaerobiosis. During anaerobic growth of S. cerevisiae, energy is derived exclusively from substrate-level phosphorylation in glycolysis. In such cells, the reoxidation of NADH formed during glycolysis does not occur through the respiratory chain that transfers the reducing equivalents to oxygen. Therefore, maintaining a redox balance is important for continuous operation of the catabolic reaction during anaerobiosis. The soluble fumarate reductase from yeast requires FADH2 , FMNH2 or reduced ribo£avin as an electron donor. The reduced equivalents of the reduced £avin nucleotides are probably supplied by NADH. In the present paper, we examined the e¡ect of arti¢cial electron acceptors, which oxidize non-enzymatically cellular NADH to NADþ , on anaerobic growth of cells containing deletion mutations in fumarate reductase. We found that the addition of oxidized
0378-1097 / 02 / $22.00 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 9 1 8 - 7
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forms of methylene blue or phenazine methosulfate overcame the inhibition of anaerobic growth of the disruptant of both the FRDS and OSM1 genes. From the above results we concluded that both fumarate reductases participate in the reoxidation of NADH generated by anaerobic metabolism.
2. Materials and methods 2.1. Strains The S. cerevisiae strains used in this study are listed in Table 1. 2.2. Media and growth conditions The disruptants of fumarate reductase genes were cultured in a chemically de¢ned medium, which contained 0.125% yeast nitrogen base (without amino acids and ammonium sulfate) (Difco), 5% glucose, 1.25% ammonium sulfate, and appropriate amounts of required amino acids. Strain DSDHH was grown in YPD medium containing 2% bactopeptone, 1% yeast extract, and 4% glucose. For anaerobic cultivation, ergosterol (0.025 g l31 ) and Tween 80 (1.05 g l31 ) were added to the medium. Other conditions of cultivation were as described previously [2]. To prepare agar plates, 1.5% Difco bacto agar was added to the de¢ned medium. The agar plates were incubated at 30‡C using AnaerobicAnaero Pack for Cell (Mitsubishi Gas Chemical, Japan) in an anaerobic chamber. 2.3. Assays of cell growth and oxidized methylene blue The concentration of oxidized methylene blue in the supernatant obtained after centrifugation of culture and cell growth was determined by measuring absorbance at 600 nm and 660 nm, respectively, using a spectrophotometer V-520 (Jasco, Japan). 2.4. Determination of succinate in culture broth The cultures collected during the exponential growth phase and stationary phase were centrifuged at 4000Ug for 20 min at 4‡C. The resulting supernatant was used to
determine succinate levels. Succinate was assayed using an HPLC organic acid analysis system (Jasco, Japan). 2.5. Fractionation of mitochondria Strain DSDHH was grown aerobically as described above. Mitochondria were prepared and fractionated as described previously [11,12]. The purity of the obtained fraction was judged from the activity of marker enzymes. The enzymatic activity was assayed for myokinase [13], citrate synthase [14], malate dehydrogenase [15], cytochrome c oxidase [16], and kynurenine hydroxylase [17]. Fumarate reductase activity was assayed as described previously [2]. Protein was assayed according to the method of Bradford [18] using a commercial kit (Pierce) with bovine albumin as a standard. 2.6. Chemicals Methylene blue was purchased from Kanto Chemical Co., Inc ; phenazine methosulfate from Sigma ; and luciferase and NAD(P)H:FMN oxidoreductase from Roche Diagnostics GmbH. All other chemicals were of reagent grade.
3. Results and discussion Under anaerobic conditions, strain DFRDOSM, which was simultaneously disrupted for both the FRDS and OSM1 genes, did not grow in the chemically de¢ned agar medium (Fig. 1B). Under such conditions, strain DOSM deleted for the OSM1 gene grew poorly, while strain DFRDS deleted for the FRDS gene as well as the wild-type strain DBY747 grew well (B). These results are consistent with those of previous studies using a YPD medium consisting of rich nutrients [9]. The addition of 2 mM methylene blue (C) or 5 mM phenazine methosulfate (D) to the agar medium fully restored anaerobic growth of strain DFRDOSM. Under aerobic cultivation, strain DBY747 and all the disruptants of fumarate reductase genes grew in the agar medium without any electron acceptors (A) or with 2 mM methylene blue or 5 mM phenazine methosulfate (data not shown). Strain DFRDOSM was anaerobically cultured in the
Table 1 S. cerevisiae strains used in this study Strain
Genotype
DBY747 DFRDS DOSM DFRDOSM DSDHH
K, K, K, K, K,
a
Source
his3-v1,leu2-3,112,trp1-289a,ura3-52 his3-v1,leu2-3,112,trp1-289a,FRDS : :URA3 his3-v1,leu2-3,112,trp1-289a,OSM1: :URA3 his3-v1,leu2-3,112,trp1-289a,FRDS : :URA3, OSM1: :THAr SDH1: :HIS3,leu2-3,112,trp-289a,ura3-52
The Yeast Genetic Stock Center.
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YGSCa [9] [9] [9] [10]
K. Enomoto et al. / FEMS Microbiology Letters 215 (2002) 103^108
chemically de¢ned liquid medium containing various concentrations of methylene blue. In the presence of 0.01 mM and 0.04 mM methylene blue, DFRDOSM grew exponentially until the oxidized methylene blue was completely exhausted (40 h); thereafter, the cell density was maintained at constant levels until 70 h (Fig. 2). The maximum cell density depended on the concentration of methylene blue added. However, the cell density was not proportional to the concentration of methylene blue. It is known that the arti¢cial electron acceptor methylene blue, oxidizes cellular NADH to NADþ [19]. Phenazine methosulfate also oxidizes NADH to NADþ in vitro and is used as a redox indicator for detection of the dehydrogenase reaction. Accordingly, fumarate reductase isoenzymes FRDS1 and FRDS2 allow for reoxidation of cellular NADH generated by anaerobic metabolism.
105
Table 2 Production of succinate by disruptants during anaerobiosis
DBY747 DFRDS DOSM DFRDOSM
Without methylene blue (mmol l31 )
0.08 mM methylene blue (mmol l31 )
0.61 0.60 0.60 ^
0.48 0.51 0.63 0.07
Strains DBY747, DFRDS, DOSM, and DFRDOSM were anaerobically cultured in chemically de¢ned liquid medium with or without 0.08 mM methylene blue. The culture was withdrawn at 70 h in the stationary phase. In all the cultivations, the oxidized methylene blue was completely exhausted at 70 h. The concentration of succinate in the supernatant was determined and expressed as mmol l31 .
Fig. 1. The e¡ect of arti¢cial electron acceptors on growth of disruptants under anaerobic conditions. Strains DBY747 (1), DFRDOSM (2), DOSM (3), and DFRDS (4) were aerobically cultured at 30‡C for 5 days in chemically de¢ned agar medium (A). Under anaerobic conditions, the strains were cultured without electron acceptors (B) or with 2 mM methylene blue (C) or 5 mM phenazine methosulfate (D).
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Fig. 2. The e¡ects of various concentrations of oxidized methylene blue on anaerobic growth of strain DFRDOSM. Strain DFRDOSM was cultured at 30‡C in chemically de¢ned liquid medium with 0 mM (E), 0.01 mM (O), 0.04 mM (U) and 0.08 mM (a) of methylene blue. Cell density (A) during cultivation was determined by measuring absorbance at 660 nm. The concentration of oxidized methylene blue (B) was determined by measuring absorbance at 600 nm.
The production of succinate by fumarate reductase disruptants during anaerobic growth in the presence or absence of methylene blue was studied. In the stationary phase, succinate production by strain DFRDOSM was markedly decreased, corresponding to 21% of that of strain DBY747 (Table 2). However, the production in strains DFRDS and DOSM was unchanged from that in the DBY747 strain. These results indicate that, under anaerobic conditions, succinate is produced by the joint activity of the two fumarate reductase isoenzymes, FRDS1 and FRDS2. The metabolism of S. cerevisiae as well as other eukaryotic cells cannot be understood without considering metabolic compartmentalization. FRDS2 and FRDS1 are known to exist in promitochondria and in the cytosol, respectively [4]. We further studied the detailed distribution of FRDS2 in mitochondria obtained from aerobically grown cells. We did not use anaerobically grown cells possessing mitochondria-like particles, which have been
designated promitochondria [20]. The reason for this is that promitochondria lack respiratory enzymes used as markers of fractionation [20]. To avoid interference by mitochondrial succinate dehydrogenase, which catalyzes not only the succinate oxidation but also the fumarate reduction, strain DSDHH deleted for the SDH1 gene encoding the £avoprotein subunit of succinate dehydrogenase [21] was utilized for determining the localization of FRDS2. Mitochondria were fractionated into three fractions: membrane (inner and outer membranes), intermembrane space, and matrix. The activities of fumarate reductase and marker enzymes in each fraction were determined. As shown in Fig. 3, a large portion of fumarate reductase activity was located in the matrix of the mitochondria. Since mitochondria are not permeable to pyridine nucleotides, including NADH, some device must exist to allow for the reoxidation of the reduced nucleotides in the compartment where they are generated. In aerobically
Fig. 3. Intramitochondrial localization of FRDS2. Strain DSDHH was grown aerobically in YPD medium. Marker enzymes used to judge the purity of the obtained fraction were kynurenine hydroxylase (KHY), cytochrome c oxidase (COX), myokinase (MKI), citrate synthase (CSY), and malate dehydrogenase (MDH). The abscissa denotes the percentages of total activity in mitochondria.
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grown cells, the excess NADH produced in the assimilation process can be reoxidized by mitochondrial respiration. However, under anaerobic conditions, respiration is inoperative. In anaerobic growth, a method for continuously regenerating the NADþ needed for glycolysis is required, because the cellular pool of pyridine nucleotide coenzymes is limited. In the glycolytic pathway, the reduction of NADþ in the glyceraldehyde-3-phosphate dehydrogenase reaction is stoichiometrically linked to the oxidation of NADH in the alcohol dehydrogenase reaction. Therefore, ethanol formation cannot account for the reoxidation of assimilatory NADH. Several reaction processes can perform this role of regenerating NADþ . As described previously, promitochondria lack an integrated electron transfer chain, a functional oxidative phosphorylation system, and several respiratory enzymes [20]. In such cells, therefore, the citrate cycle does not appear to proceed in a cyclic manner. Nissen et al. [22] showed that a large part of the mitochondrial NADH is produced from the synthesis of 2-oxoglutarate, which is a precursor of glutamate. In the present study, we found that the inhibition of the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was overcome by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NADþ . We conclude that in anaerobically grown cells, FRDS2 in the promitochondria matrix and FRDS1 in the cytosol are involved in the reoxidation of excess NADH in each compartment. Soluble fumarate reductase from yeast requires FADH2 and FMNH2 , but not NADH, as electron donors for fumarate reduction. Thus, the enzyme catalyzing the conversion of the oxidized £avin nucleotides by NADH to the reduced forms is required for the reoxidation of NADH by fumarate reductase. At the present time, the enzyme in S. cerevisiae possessing such characteristics remains to be identi¢ed. Other ways to maintain the redox balance during anaerobiosis are known in S. cerevisiae. Cytosolic GPD2, which is one of the glycerol-3-phosphate dehydrogenase isoenzymes, is important for the reoxidation of the cytosolic NADH that migrates from mitochondria [23]. Under anaerobic conditions, a GPD2-deleted mutant grows more slowly and accumulates less glycerol than the wild-type strain. Also, mitochondrial ADH3, one of the alcohol dehydrogenases, may play a key role in the reoxidation of NADH in anaerobically grown cells [24,25]. The anaerobic growth of the ADH3-deletion mutant was slower than that of the wild strain. The ethanol produced in mitochondria by ADH3 would be expected to di¡use into the cytosol, resulting in the transport of excess intramitochondrial NADH to the cytosol [26]. Fumarate reductase, glycerol3-phosphate dehydrogenase, and alcohol dehydrogenase may cooperatively function in maintaining the redox balance during anaerobiosis.
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