INTRODUCTION

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As in other eukaryotes, respiratory dissimilation of sugars by Saccharomyces cerevisiae leads to the reduction of NAD⁺ to NADH in separate cellular compartments. Cytosolic NADH is produced by the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, as well as in assimilatory reactions (1, 30). In the mitochondrial matrix, NADH is formed by the tricarboxylic acid cycle and the pyruvate-dehydrogenase complex. Under anaerobic conditions, glucose dissimilation occurs exclusively via alcoholic fermentation, which is a redox-neutral process. The additional NADH originating from biomass production can be reoxidized via glycerol production (30). Under aerobic conditions, glycerol production is not necessary, because the reoxidation of cytosolic NADH can be coupled to the mitochondrial respiratory chain.

Although the outer mitochondrial membrane is permeable to NADH (17), the inner membrane is not (35). Therefore, coupling of NADH reoxidation to the respiratory chain has to occur on both sides of the mitochondrial inner membrane. In plant mitochondria, cytosolic NADH can be oxidized either by an external NADH dehydrogenase or by a redox shuttle (14), whereas in the mitochondrial matrix, NADH can be oxidized by the proton-translocating complex I or by an alternative, nontranslocating, internal NADH dehydrogenase (9). Mammalian mitochondria lack an external NADH dehydrogenase and therefore rely on redox shuttle mechanisms to couple the oxidation of cytosolic NADH to complex I (5).

In S. cerevisiae, the NADH in the mitochondrial matrix can be oxidized by an NADH:ubiquinone oxidoreductase (35). This enzyme is located in the inner mitochondrial membrane, and its active site faces the mitochondrial matrix. In contrast to the classical complex I of higher eukaryotes, the S. cerevisiae internal NADH dehydrogenase (Ndi1p [Fig. 1]) does not translocate protons (7, 22). The enzyme is encoded by a single nuclear gene, NDI1 (8). Mitochondria isolated from ndi1 null mutants do not oxidize intramitochondrial NADH (20).

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Overview of possible mechanisms of oxidation of cytosolic NADH by mitochondria of S. cerevisiae. Abbreviations: R[H2], reduced metabolite; R, oxidized metabolite; Gpd, cytosolic glycerol-3-phosphate dehydrogenase; Gut2, membrane-bound mitochondrial glycerol-3-phosphate dehydrogenase; Nde, external NADH dehydrogenase; Ndi, internal NADH dehydrogenase; Q, ubiquinon pool; bc1, cytochrome bc1 complex; cox, cytochrome c oxidase.

Several mechanisms have been proposed for reoxidation of cytosolic NADH by S. cerevisiae mitochondria (Fig. 1). First, it can be oxidized by the external NADH dehydrogenases Nde1p and Nde2p. These dehydrogenases are homologous to Ndi1p, but their active sites face the lumen between the membranes (19, 29). Similar to the internal NADH dehydrogenase, they directly couple the oxidation of NADH to the respiratory chain. Another mechanism for oxidation of cytosolic NADH is the glycerol-3-phosphate shuttle (Fig. 1), all of whose essential enzymes are present in S. cerevisiae (28). A recent study indicates that although the glycerol-3-phosphate shuttle is functional in this yeast, it is not essential for respiratory growth on ethanol (16). Other proposed shuttles include the ethanol-acetaldehyde shuttle (35), the malate-oxaloacetate shuttle (3), and the malate-aspartate shuttle (3). All these shuttles operate according to a similar principle (Fig. 1) but with different substrates. However, it is unknown whether these shuttles operate in vivo in S. cerevisiae.

As discussed above, the relative importance of the various proposed systems for respiratory oxidation of cytosolic NADH by S. cerevisiae mitochondria is unclear. Since these systems are coupling the oxidation of cytosolic NADH to the respiratory chain, their function must be studied under respiratory growth conditions. However, S. cerevisiae has a strong tendency toward alcoholic fermentation. Even under fully aerobic conditions, a mixed respirofermentative metabolism is observed when the sugar concentration in the growth medium exceeds a threshold value (typically ca. 1 mM [34]) or when the specific growth rate is high (usually higher than two-thirds of the maximal growth rate [25, 26]). Instead of studying the NADH-oxidizing systems in shake flask cultures, in which sugar metabolism is predominantly fermentative (15), it is essential to use aerobic, glucose-limited chemostat cultures for this purpose. In glucose-limited chemostat cultures the glucose concentration is very low and the specific growth rate, and hence the rate of glycolysis, can be controlled by varying the dilution rate (25). In this way, glucose catabolite repression of respiratory enzymes (11) and alcoholic fermentation can be avoided.

The aim of the present study is to assess the physiological significance of the external NADH dehydrogenases, the glycerol-3-phosphate shuttle, and possible other mechanisms in the oxidation of cytosolic NADH by S. cerevisiae mitochondria. To investigate the relative importance of the external NADH dehydrogenases and the glycerol-3-phosphate shuttle, the physiology of an nde1 nde2 and a gut2 mutant was studied while varying the specific growth rate of the culture and thereby the turnover of cytosolic NADH. Furthermore, to investigate whether other enzymes are involved in the respiration of cytosolic NADH, the physiology of an nde1 nde2 gut2 mutant was studied.

	MATERIALS AND METHODS

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Yeast strains and maintenance. The S. cerevisiae strains used in this study (Table 1) were maintained by cultivating them in shake flasks on complex medium containing 1% (wt/vol) Bacto yeast extract, 2% (wt/vol) peptone, and 2% (wt/vol) glucose (YPD medium). When stationary phase was reached, 30% (vol/vol) sterile glycerol was added and 2-ml aliquots were stored in sterile vials at 80°C. These stock cultures were used subsequently as the inoculum for precultures from which all experiments were started.

Construction of null mutants. Standard techniques and media for genetic manipulation of S. cerevisiae were used (2). Deletion in GUT2 (YIL155c) was obtained by the short flanking homology method (36) using plasmid pUG6 as the template (12). PCR primers were used for amplification of the loxP-kanMX-loxP cassette and for verification of the correct gene deletion (Table 2). PCR amplifications, yeast transformation, and verification of the correct gene deletion as well as determination of the mating type were carried out as described by Luttik et al. (19). To obtain a strain with GUT2, NDE1 (YMR145c) and NDE2 (YDL085w) deleted, strains were crossed as follows. Strain CEN.PK225-1B (gut2) was crossed with CEN.PK152 (nde1) and was also crossed with CEN.PK163 (nde2), resulting in strains CEN.PK239-1C (nde1 gut2) and CEN.PK240-1C (nde2 gut2), respectively. These double-mutant strains were subsequently crossed again to construct strain CEN.PK263-5D (nde1 nde2 gut2). In all cases, strains were analyzed by tetrad analysis and spores showing the nonparental ditype for the KanMX marker were subsequently verified by diagnostic PCR to confirm the correct deletion of the corresponding genes.

Isolation of mitochondria. The procedure for the isolation of mitochondria, which involved enzymatic degradation of the cell wall, controlled lysis of the spheroplasts, and harvesting of mitochondria by differential centrifugation, has been described previously (19). The protein content of mitochondrial preparations was determined by the method of Lowry et al. (18) with bovine serum albumin (fatty acid free; Sigma) as a standard. Protein contents were corrected for the bovine serum albumin added to the buffer in which the mitochondria were resuspended.

Oxygen consumption by isolated mitochondria. Specific rates of oxygen consumption were measured in a 4-ml thermostatted vessel (30°C) with a Clark-type oxygen electrode. Experiments were performed in a buffer (pH 7.0) consisting of 25 mM potassium phosphate, 5 mM MgCl₂, and 0.65 M sorbitol. Substrates were added at a final concentration of 5 mM. Since commercially available NADH is contaminated with ethanol (24), pure NADH was generated in situ using Bacillus megaterium glucose dehydrogenase (19). Each substrate was tested at two different dilutions of the mitochondrial preparation. Oxygen consumption rates were calculated based on a dissolved-oxygen concentration of 236 µM in air-saturated water at 30°C. Respiratory-control values were determined by adding 0.25 mM ADP (4).

Chemostat cultivation. S. cerevisiae wild-type and mutant strains were cultivated in a laboratory fermentor as described previously (19). This reference also describes the gas analysis and the determination of the culture dry weight. The culture medium used was a defined mineral medium, with vitamins prepared as described by Verduyn et al. (33), containing 7.5 g of glucose per liter as the carbon source. Dilution rate ranges were started a dilution rate of 0.05 h¹. After a steady state was reached, the dilution rate was increased in steps of 0.05 h¹ or, when close to the point where ethanol started to appear, in steps of 0.025 h¹. For each dilution rate, a steady state was reached before the dilution rate was increased further.

Metabolite measurements. The glucose concentration in the medium was determined by enzymatic analysis with a hexokinase and glucose-6-phosphate dehydrogenase kit (Boehringer Mannheim). The concentrations of glycerol, ethanol, and acetate in culture supernatants were determined by high-pressure liquid chromatography and confirmed by enzymatic analysis (27). The protein content of whole cells was determined by a modified biuret method (32). The calculated carbon recoveries were between 95 and 103% for all cultures, with an assumed carbon content of biomass of 48%.

	RESULTS

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Oxygen consumption by isolated mitochondria. To confirm the absence of the gene products (and any isoenzymes) in the deletion mutants, substrate-dependent oxygen consumption by isolated mitochondria was measured in wild-type S. cerevisiae and in isogenic gut2, nde1 nde2, and nde1 nde2 gut2 strains (Table 3). External NADH dehydrogenase activity was completely abolished in the nde1 nde2, and nde1 nde2 gut2 mutants. An earlier report (29) that mitochondria of an nde1 nde2 mutant exhibited residual oxidation of NADH can be explained by the use of commercial NADH (19). This is contaminated with ethanol (24), which is readily oxidized by S. cerevisiae mitochondria. Glycerol-3-phosphate dehydrogenase activity was completely abolished in gut2 mutants.

Physiology of wild-type S. cerevisiae in aerobic, glucose-limited chemostat cultures. The physiological consequences of the different deletions were studied by growing wild-type and mutant S. cerevisiae strains in aerobic, glucose-limited chemostats at dilution rates varying from 0.05 to 0.38 h¹ (Fig. 2). If a chemostat culture is at steady state, the dilution rate is by definition equal to the specific growth rate. At specific growth rates below 0.30 h¹, the wild-type strain CEN.PK113-7D (Fig. 2A) had a high biomass yield (0.5 g of biomass g of glucose¹) and produced neither ethanol nor glycerol. Completely respiratory growth was also evident from the respiratory quotient (the specific CO₂ production rate, q_CO2, divided by the specific O₂ consumption rate, q_O2), which was 1.0 at specific growth rates below 0.30 h¹ (Fig. 3A). Above a dilution rate of 0.30 h¹, ethanol was produced and the respiratory quotient increased to values above 1.0 as the q_CO2 increased and the q_O2 decreased (Fig. 2A and 3A). Concomitantly, the biomass yield decreased sharply. Glycerol production was observed only at the highest dilution rate applied. The highest dilution rate at which wild-type cultures reached a steady state was 0.38 h¹. Above this dilution rate, the wild-type strain washed out of the chemostat.

View larger version (31K): [in this window] [in a new window]   FIG. 2.   Effects of dilution rate in an aerobic, glucose-limited chemostat culture on biomass yield (YXS) and specific production rate of ethanol and glycerol (respectively qethanol and qglycerol) for wild-type S. cerevisiae CEN.PK113-7D (data taken from reference 31) (A), gut2 mutant CEN.PK225-2C (B), nde1 nde2 mutant CEN.PK167-2B (C), and nde1 nde2 gut2 mutant CEN.PK263-5D (D). Apparently filled-in symbols are a result of overlapping data points from independent chemostat cultures.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Effects of dilution rate in an aerobic, glucose-limited chemostat culture on the specific production rate of CO2 (qCO2) and the specific consumption rate of O2 (qO2) for wild-type S. cerevisiae CEN.PK113-7D (data taken from reference 31) (A), gut2 mutant CEN.PK225-2C (B), nde1 nde2 mutant CEN.PK167-2B (C), and nde1 nde2 gut2 mutant CEN.PK263-5D (D). Apparently filled-in symbols are a result of overlapping data points from independent chemostat cultures.

Physiology of a gut2 mutant. In S. cerevisiae the glycerol-3-phosphate shuttle can, in principle, couple the oxidation of cytosolic NADH to the respiratory chain. To test whether its contribution is essential, the GUT2 gene, encoding the mitochondrial membrane-bound glycerol-3-phosphate dehydrogenase, was deleted. When the gut2 mutant was cultivated over a range of dilution rates, the measured metabolite production rates were essentially the same as those of the wild type (Fig. 2B and 3B). Aerobic fermentation set in at the same dilution rate as in the wild type (0.30 h¹), but washout of biomass occurred at a lower dilution rate (0.33 versus 0.38 h¹). Apparently, elimination of the mitochondrial membrane-bound glycerol-3-phosphate dehydrogenase hardly affected the respiratory capacity during aerobic glucose-limited growth. Thus, even if the glycerol-3-phosphate shuttle were active in a wild-type strain, in the mutant its contribution to the oxidation of cytosolic NADH could be taken over completely by other NADH-oxidizing systems.

Physiology of an nde1 nde2 mutant. Cytosolic NADH can also be oxidized by the external NDE1- and NDE2-encoded NADH dehydrogenases. In contrast to the gut2 mutant, the physiology of an nde1 nde2 mutant in aerobic, glucose-limited chemostat cultures differed substantially from that of the wild-type strain (Fig. 2C and 3C). The nde1 nde2 mutant produced glycerol at all specific growth rates tested, albeit in small quantities at low dilution rates, whereas the wild-type strain produced glycerol only at a dilution rate of 0.38 h¹. No ethanol production was observed up to a dilution rate of 0.23 h¹. Above this dilution rate, ethanol was produced and, concomitantly, the specific glycerol production rate decreased, suggesting a decreased need for a cytosolic redox sink under these conditions. At the highest applied dilution rate, the glycerol production increased again. The respiratory quotient was 1.0 at low dilution rates but increased as production of glycerol and ethanol became apparent (Fig. 3C). Washout of biomass occurred at dilution rates above 0.33 h¹. At low dilution rates, the biomass yield was almost as high as that of the wild type, but it decreased when glycerol and ethanol were being produced. Apparently, other NADH-oxidizing systems, such as the glycerol-3-phosphate shuttle, could not completely replace the external NADH dehydrogenases but had a sufficiently high capacity to prevent alcoholic fermentation at low dilution rates.

Physiology of an nde1 nde2 gut2 mutant. To investigate whether, in addition to the glycerol-3-phosphate shuttle and the external NADH dehydrogenases, other enzymes contribute to the respiration of cytosolic NADH, an nde1 nde2 gut2 mutant was grown in aerobic, glucose-limited chemostat cultures. At dilution rates between 0.05 and 0.15 h¹ (Fig. 2D and 3D), this mutant produced no ethanol but its specific glycerol production rate was high. The biomass yield of the nde1 nde2 gut2 mutant (0.3 g g¹) was significantly lower than that of the wild type (0.5 g g¹).

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	DISCUSSION

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Physiological significance of Nde1/2p and Gut2p. The production of glycerol by aerobic, glucose-limited chemostat cultures of the nde1 nde2 mutant (Fig. 2C) is indicative of redox stress. This observation shows that external NADH dehydrogenase is involved in respiratory glucose dissimilation by wild-type S. cerevisiae. A gut2 mutant did not exhibit such a phenotype (Fig. 2B). Apparently, either this system is less important in wild-type cells or its role can be taken over by the external NADH dehydrogenases and/or alternative systems. The observation that respiratory growth on glucose was more severely impaired in an nde1 nde2 gut2 mutant than in an nde1 nde2 mutant confirms the earlier conclusion that the glycerol-3-phosphate shuttle can operate in S. cerevisiae (16).

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Alternative mechanisms for oxidation of cytosolic NADH. One explanation for the lack of ethanol production by the nde1 nde2 gut2 mutant at low dilution rates is cytosolic conversion of glucose to equimolar amounts of pyruvate and glycerol (Fig. 4A). This results in a closed cytosolic redox balance, because each molecule of NADH formed in the production of pyruvate is reoxidized in the production of glycerol from glucose. Pyruvate can be oxidized further in the mitochondria, leading to the production of 1 mol of glycerol and 3 mol of CO₂ for each dissimilated mole of glucose. At a dilution rate of 0.1 h¹, the specific production rate of glycerol in the nde1 nde2 gut2 mutant was 1.2 mmol of glycerol g of biomass¹ h¹. The amount of glycerol formed according to the scheme depicted in Fig. 4A should correspond to the sum of the NADH formed during biomass synthesis and the pyruvate produced in glycolysis, and it can be calculated as follows. Assuming a constant biomass composition, the rate of glycerol production as a result of biomass formation in the mutant equals the value observed in anaerobic wild-type cultures: ca. 0.9 mmol of glycerol g of biomass¹ h¹ (32, 37). The amount of pyruvate formed during glucose catabolism in the mutant can be approximated from the rate of CO₂ production, since mitochondrial oxidation of 1 mol of pyruvate yields 3 mol of CO₂. The rate of CO₂ production by the nde1 nde2 gut2 mutant at a dilution rate of 0.1 h¹ equaled 3.9 mmol g¹ h¹ (Fig. 3D), corresponding to approximately 1.3 mmol of pyruvate g¹ h¹. Thus, the total rate of glycerol production expected according to Fig. 4A would amount to 0.9 + 1.3 = 2.2 mmol g¹ h¹. Since the observed value was only 1.2 mmol g¹ h¹, glycerol production cannot be solely responsible for the oxidation of cytosolic NADH in the nde1 nde2 gut2 mutant. This signifies that in addition to the external NADH dehydrogenases and the glycerol-3-phosphate shuttle, at least one other mechanism must exist which can oxidize cytosolic NADH.

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Schematic representation of proposed metabolic pathways for the oxidation of cytosolic NADH in an S. cerevisiae nde1 nde2 gut2 mutant, grown in an aerobic, glucose-limited chemostat culture at low dilution rates. (A) Conversion of dissimilatory glucose into equimolar amounts of pyruvate and glycerol; (B) consumption of ethanol produced in cytosol by mitochondria; (C) ethanol-acetaldehyde shuttle.

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Significance of mitochondrial redox shuttles. In vivo, mitochondrial ethanol consumption (Fig. 4B) and the ethanol-acetaldehyde shuttle (Fig. 4C) may occur simultaneously: in the latter mechanism, acetaldehyde returns to the cytosol, while in the former, it is oxidized to acetate inside the mitochondria. The contribution of the two mechanisms may depend on activities of key enzymes (in particular mitochondrial acetaldehyde dehydrogenase, which withdraws acetaldehyde from the shuttle cycle), as well as on the intracellular concentrations of acetaldehyde and ethanol. It is conceivable that at low dilution rates (0.15 h¹), reoxidation of glycolytic NADH in the nde1 nde2 gut2 mutant involves ethanol consumption by the mitochondria, whereas at dilution rates of 0.20 h¹, where ethanol appears outside the cells, the yeast gradually shifts to the ethanol-acetaldehyde shuttle. The reduced glycerol production by the nde1 nde2 gut2 mutant at dilution rates above 0.15 h¹, which coincided with an increased rate of ethanol production (data not shown), is consistent with a need for the presence of ethanol to obtain a functional shuttle. The nde1 nde2 mutant displayed similar behavior at dilution rates above 0.25 h¹ (Fig. 2C).

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