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Involvement of mitochondrial aldehyde dehydrogenase ALD5 in maintenance of the mitochondrial electron transport chain in Saccharomyces cerevisiae

Osamu Kurita, Yoshio Nishida
DOI: http://dx.doi.org/10.1111/j.1574-6968.1999.tb08856.x 281-287 First published online: 1 December 1999


The physiological role of mitochondrial aldehyde dehydrogenase (ALD5) was investigated by analysis of the ald5 mutant (AKD321) in Saccharomyces cerevisiae. K+-activated ALDH activity of the ald5 mutant was about 80% of the wild-type in the mitochondrial fraction, while the respiratory activity of the ald5 mutant was greatly reduced. Cytochrome content was also reduced in the ald5 mutant. Enzymatic analysis revealed that the alcohol dehydrogenase activity of the ald5 mutant was higher than that of the wild-type, while glycerol 3-phosphate dehydrogenase activity was the same in the two strains. Ethanol as a carbon source or addition of 1 M NaCl with glucose as the carbon source in the growth medium increased β-galactosidase activity from an ALD5-lacZ fusion. Overexpression of another mitochondrial ALDH gene (ALD7) had no effect on increasing respiratory function of the ald5 mutant, but showed improved growth on ethanol. These observations show that mitochondrial ALD5 plays a role in regulation or biosynthesis of electron transport chain components.

  • Saccharomyces cerevisiae
  • Aldehyde dehydrogenase
  • Mitochondrion
  • Electron transport chain

1 Introduction

Elucidation of the complete sequence of the genome of the yeast Saccharomyces cerevisiae has revealed the existence of several aldehyde dehydrogenase isozymes. The associated ALDH genes have been preliminarily identified and characterized. YMR170c (ALD2) on chromosome XIII was shown to be induced by osmotic stress [1]. YPL061w (ALD6) on chromosome XVI, encoding a cytosolic, Mg2+-activated acetaldehyde dehydrogenase, was found to be involved in the production of acetate from which cytosolic acetyl-CoA was synthesized [2]. YOR374w (ALD7) on chromosome XV, encoding a mitochondrial K+-activated ALDH, was found to be involved in the oxidative metabolism of ethanol [3,4]. YER073w (ALD5) on chromosome V was shown to be a mitochondrial enzyme with increased activity in the presence of K+ ions [5].

We previously isolated ald mutants which were partially deficient in ALDHs and suggested that the ALDH isozymes played a role in ethanol stress and salt stress [6]. Our interest has been to clarify individual physiological characteristics of the various ALDH isozymes. Mitochondrial ALD5 has not yet been defined. Moreover, yeast ALD5 is the homologue of the human gene associated with acute alcohol intoxication [7]. We have used an ald5 mutant to investigate the physiological role of this gene. Here, we report data that ALD5 is involved in regulation or maintenance of oxidative phosphorylation in S. cerevisiae.

2 Materials and methods

2.1 Yeast strains and media

S. cerevisiae DBY746 strain (αhis3 leu2-3 leu2-112 trp1-289 ura3-52; The Yeast Genetic Stock Center) was the host in which ALD5 was disrupted. AKD321 (αhis3 leu2-3 leu2-112 trp1-289 ura3-52 ald5::LEU2) is a haploid strain from which the coding region of YER073w/ALD5 has been disrupted by insertion. Yeast cells were routinely grown in YPD medium (2% glucose, 1% yeast extract, 2% polypeptone). YPE medium was similar to YPD medium, except that 3% ethanol replaced glucose. YNB medium contained 2% glucose, 0.67% yeast nitrogen base without amino acids (Difco), and supplements (40 mg l−1) to satisfy auxotrophic requirements for growth. All solid media contained 2% agar.

2.2 PCR cloning of S. cerevisiae ALD5 and ALD7 genes

Genomic DNA of DBY746 was prepared by standard methods [8]. PCR was used to generate the coding regions of ALD5 and ALD7. The two primers for ALD5 were 5′-GATCAGGTCCTTATGATGACAGA-3′ corresponding to nucleotides −952 to −930 (forward primer) and 5′-GCCAAACCGTATCTGACTAATCT-3′ complementary to nucleotides +3048 to +3026 (reverse primer). The 4.0-kb PCR product was blunt-ended by T4 DNA polymerase, phosphorylated by T4 polynucleotide kinase and then ligated to the SmaI site of integrating vector pRS406 (designated PRS406-ALD5). The two primers for ALD7 were 5′-GGGATAGGATCCTCCAGGATGA-3′ (BamHI site underlined) corresponding to nucleotides −1260 to −1239 (forward primer) and 5′-AAGGTCGACATTGCTTTGCCATGTGCCA-3′ (SalI site underlined) complementary to nucleotides +2288 to +2260 (reverse primer). The 3.6-kb PCR product was digested with BamHI and SalI and was cloned into the BamHI/SalI sites of multi-copy vector pRS426 [9]. The resultant plasmid was called pRS426-ALD7.

2.3 Gene disruption

A 2.2-kb SalI-XhoI fragment carrying the selectable LEU2 gene was blunt-ended and was cloned into the BstEII site of PRS406-ALD5 whose ends had been blunt-ended previously. The BstEII site corresponded to nucleotides +1054 in the ALD5 sequence. The resulting plasmid (pALD5-D1) was digested with HindIII (771 bp upstream of the BstEII site) and KpnI (1329 bp downstream of the BstEII site). This fragment was used to transform the recipient strain DBY746 for one-step gene disruption [10]. Yeast transformation was performed by the method of Ito et al. as described previously [11]. The disruption was confirmed by PCR and Southern blot analysis.

2.4 ALD5-lacZ fusion

The 675-bp EcoRV-HindIII fragment of ALD5, which consisted of the promoter region and the first 51 bp of the reading frame, was fused to lacZ in plasmid YEp365 [12] cut with SmaI and HindIII. Yeast cells carrying the lac′Z fusion plasmid were grown to the late exponential phase in YPD, YPD+1 M NaCl (supplement containing 1 M NaCl in YPD), or YPE medium at 30°C with shaking. Preparation of cell-free extracts and β-galactosidase assays were performed [13].

2.5 Preparation of cell-free extracts and mitochondrial purification and fractionation

Late exponential phase cells were harvested by centrifugation and washed twice with distilled water. The yeast cells were suspended in 0.1 M Tris-HCl (pH 8.0), 1 mM EDTA, and 1 mM dithiothreitol and disrupted with glass beads in a MSK cell homogenizer (B. Braun, Melsungen). The extract was centrifuged at 12 000×g for 15 min, and the supernatant was used as a cell-free extract for enzyme assays.

Mitochondria were prepared by a spheroplast method as described previously [6] and were disrupted in the cold phase by sonication for 1 min at 300 W. The disrupted mitochondria were centrifuged at 150 000×g for 90 min, and the supernatant was used as a sample for enzyme assays.

2.6 Enzyme assay

K+-activated aldehyde dehydrogenase was assayed by the increase of absorbance at 340 nm at 25°C. The reaction mixture consisted of 50 mM Tris-HCl (pH 8.0), 2 mM acetaldehyde, 1 mM DTT, 0.1 M KCl and 1 mM NAD+. Alcohol dehydrogenase activity was measured spectrophotometrically as described previously [6]. Glycerol 3-phosphate dehydrogenase was assayed with 50 mM triethanolamine buffer pH 7.9, 0.5% bovine serum albumin, 0.18 mM NADH, and 1 mM dihydroxyacetone phosphate. Protein concentration was measured by the method of Lowry et al. with bovine serum albumin as a standard [14].

2.7 Measurement of respiratory activity

Respiratory activity of whole cells (1–10 mg dry weight) in 3.0 ml of buffer containing 2 mM glucose and 50 mM K-PO4, pH 7.4, was measured in a Clark oxygen electrode cell at 25°C.

2.8 Cytochrome determinations

Cytochrome content was estimated by absorbance in the difference spectra between 650 nm and 500 nm. For this test, mitochondria were suspended in 3.3 M glucitol at a concentration of 1 mg ml−1 and were placed in the reference and sample cuvettes. The suspension in the sample cuvette was reduced with a few grains of sodium dithionite.

2.9 Fermentation

Batch fermentation of the yeast cells was carried out at 30°C in a B.E. Marubishi MDL300 at a working volume of 1.5 l without aeration, at a stirring speed of 300 rpm. The fermented yeast cells were precultured in 500-ml Erlenmeyer flasks containing YNB medium for 24 h at 30°C on a rotary shaker at 100 rpm. Cultures were started in YPD medium with 0.01 volume of the 24-h preculture. The supernatants of the cultures obtained after centrifugation were used for metabolite analysis. Fermentation experiments were carried out in duplicate.

2.10 Analysis

Glucose was measured with Boehringer Mannheim Biochemicals F-kits 716251. Ethanol was measured by gas-liquid chromatography with n-propanol as the internal standard [6]. Acetic acid was measured by HPLC (Japan Spectroscopic Co., Ltd., 880-Type) on a Shodex Ionpak C-811.

3 Results

3.1 Intracellular distribution of K+-activated ALDH activities

Two K+-activated ALDHs (ALD5 and ALD7) are mitochondrial in origin [35]. In this experiment, the contribution of ALD5 to total ALDH activity in mitochondria was investigated (Table 1). The activity of K+-activated ALDH in the ald5 mutant AKD321 was approximately 79% that of the wild-type. The mitochondrial fraction retained considerable K+-activated ALDH, consistent with the result of Saigal et al. [15]. Nearly the same difference was observed in the cytosolic activity (65% of the wild-type) and this was considered to be mitochondrial contamination. In addition, the mitochondrial fraction of both strains was verified as having been prepared at the same level of purity using fumarase activity as a marker for mitochondrial enzyme.

View this table:
Table 1

Intracellular distribution of K+-activated aldehyde dehydrogenase activity

StrainFractionProtein (mg fraction−1)K+-activated ALDH (mU mg−1 of protein)
DBY746 (ALD5)Cytosol902.6
AKD321 (ald5::LEU2)Cytosol891.7
  • One unit of enzyme activity is the amount of enzyme producing 1 µmol of NADH per minute. Data are the average of three determinations on each fraction, variation between triplicates was less than 15%.

3.2 Cytochrome spectra and respiratory activity

In order to examine the effect on mitochondrial function of disruption of ALD5, we investigated the cytochrome spectra of mitochondria of both strains. The ald5 mutant showed no absorption maxima of cytochromes a+a3, b, or c which were found at 605 nm, 563nm, and 554 nm, respectively (Fig. 1). The absorption spectrum of the ald5 mutant was different from petite mutants. Petite mutants lack cytochromes a+a3, b, and c1 but have normal or near normal amounts of cytochrome c [16]. Thus, mitochondrial respiration by oxidative phosphorylation might be defective in the ald5 mutant. We investigated respiratory activity to test this. The respiratory activities were 5.2 and 0.57 O2µM mg−1 dry weight min−1 for strains DBY746 and AKD321, respectively. The respiratory activity of the ald5 mutant was one order of magnitude lower in comparison to that of the wild-type.

Figure 1

Visible absorption spectra of mitochondrial cytochromes in wild-type (DBY746) and the ald5 mutant (AKD321). Cells were grown for 48 h in YPD medium. After purification and fractionation of the mitochondria, they were suspended to a concentration of 1 mg ml−1 and the difference spectra of the dithionite-reduced sample were determined with a double-beam spectrophotometer.

3.3 Enzyme assay

S. cerevisiae has the ability to ferment and respire. To investigate how fermentative ability is affected by a deficiency in mitochondrial function, we assayed alcohol dehydrogenase and glycerol 3-phosphate dehydrogenase which are known to be regulatory enzymes of main fermentation and Neuberg's second fermentation, respectively. The ADH activity of the ald5 mutant was about 2.5 times that of the wild-type, while the GPD activities of both strains were much the same (Table 2).

View this table:
Table 2

Specific activities of ADH and GPD in cell-free extracts of the strains

StrainEnzyme activities (mU mg−1 of protein)
Alcohol dehydrogenaseGlycerol 3-phosphate dehydrogenase
DBY746 (ALD5)15908.1
AKD321 (ald5::LEU2)39606.9
  • One unit of enzyme activity is the amount of enzyme producing 1 µmol of NADH or NAD per minute. Data are the average of three independent determinations.

3.4 Analysis of the promoter of the ALD5 gene

To analyze regulation of the ALD5 gene, the promoter region was fused to the lacZ reporter gene. We measured β-galactosidase activities of the transformant which was grown in YPD, YPD+1 M NaCl, and YPE media. Addition of 1 M NaCl to the YPD medium and replacement of glucose with ethanol as carbon source in the growth medium greatly increased ALD5-lacZ expression levels (5 times in YPD+1 M NaCl and 9 times in YPE compared with YPD) (Fig. 2).

Figure 2

Effect of growth medium on expression of the ALD5-lacZ fusion. β-Galactosidase activity was measured in late exponential cells grown in YPD, YPD+1 M NaCl, and YPE medium.

3.5 Effect of overexpression of ALD7 on metabolite formation in ald5 mutants

A distinction in the roles of the two mitochondrial ALDHs (ALD5 and ALD7) was investigated by observing whether overexpression of the ALD7 gene could be complementary to the disrupted ALD5 gene. The ald5 mutant showed no diauxic growth after its glucose source was exhausted. The ald5 mutant AKD327 which had been transformed with PRS426-ALD7, however, grew well on ethanol after a diauxic lag phase (Fig. 3). Besides, the mutant AKD327 produced more acetic acid after stationary phase while dissolved oxygen increased temporarily during this phase. In addition, the respiratory activity of AKD327 was much the same as AKD321 (0.74 O2µM mg−1 dry weight min−1). Plasmid stability was more than 80% during the batch fermentation (data not shown).

Figure 3

Growth, glucose consumption, dissolved oxygen, and product formation in batch cultures of the ald5 mutant and the pRS426-ALD7-transformed ald5 mutant. Growth was followed by measuring the turbidity of the culture at 660 nm. Concentrations of metabolites in culture supernatants were measured. A: Strain AKD321 (ald5::LEU2). B: Strain AKD327 (ald5::LEU2 pRS426-ALD7). Data are presented as means of duplicate experiments.

4 Discussion

The mitochondrial ALDH gene, located on chromosome V, was cloned and designated ALD5 by Wang et al. [5]. They found that this ALDH was potassium-activated and utilized both NAD and NADP as cofactors. However, the physiological role of ALD5 has not been completely elucidated. Therefore, our study examined the phenotype of a single-disrupted ALD5 strain. The K+-activated ALDH activity of the ald5 mutant was 80% of the wild-type in the mitochondrial fraction. The data suggest that ALD5 is a relatively minor enzyme, consistent with the enzyme kinetic data reported by Wang et al. [5].

In the ald5 mutant, alcohol dehydrogenase (ADH) activity was found to be increased and respiratory activity decreased. It appeared that the increase of ADH activity resulted from a decrease of energy from lowered respiration ability which was dependent on mitochondrial electron transfer. In fact, ald5 gave no characteristic absorption spectra of mitochondrial cytochromes.

Yeast cells almost completely deficient in all cytochromes were isolated by introducing two defective nuclear genes, cyd1 and cyd4, into the parent strain [17]. Normal synthesis of all cytochromes was restored in the double mutant by adding δ-aminolevulic acid to the growth medium. The first phase of heme biosynthesis is a condensation of succinyl-CoA with glycine followed by decarboxylation to form δ-aminolevulinic acid [18]. The citric acid cycle intermediate succinyl-CoA is derived from acetate's methyl C atom while atom C (4) comes from the carbonyl C atom of acetate. Our experimental data suggest that ALD5 might play a role in the biosynthesis of heme which is an essential component of the cytochromes. There was no evidence that oxidation of acetaldehyde required a functioning respiratory chain in S. cerevisiae. On the other hand, in Candida maltosa, mitochondria catalyzed acetaldehyde oxidation which was coupled to oxidative phosphorylation [19].

The result of ALD5-lacZ showed that the expression of ALD5 increased by the addition of NaCl and ethanol. The ALD5 promoter contains two putative stress elements (STRE, consensus sequences CCCCT or AGGGG) at positions −263 and −638 with respect to the translation start site [20]. Blomberg and Adler found that acetate production was enhanced during osmotic stress [21]. ALD2 and ALD6 are known to be induced by osmotic stress [1,22]. ALD5 seems to respond to osmotic stress from the point of view of mitochondrial respiration as well.

Jacobson and Bernofsky previously reported that K+-activated ALDH, which was recently designated ALD7, was repressed during the growth of S. cerevisiae on glucose and was involved in the oxidative metabolism of ethanol [4]. Consistent with this observation, increased acetate production resulted from overexpression of ALD7 after glucose depletion in the ald5 mutant AKD327 transformed with PRS426-ALD7. The temporary increase of dissolved oxygen may be caused by a defective transition from fermentation to respiration imposed by overexpression of ALD7. Certainly, recovery of growth on ethanol was observed, but respiratory activity did not show the expected ALD7 gene dosage effect in the ald5 mutant. Thus, ALD5 as well as ALD7 are engaged in the oxidation of ethanol, but the former might play a regulatory role rather than a catabolic one in mitochondrial respiration in view of its low contribution to total ALDH activity in mitochondria.


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