1 Universitätsklinikum Frankfurt, Institut für Biochemie I, D-60590 Frankfurt am Main, Federal Republic of Germany
* Present address: Universität Heidelberg, Institut für Molekulare Genetik, D-69120 Heidelberg, Federal Republic of Germany
Author for correspondence (e-mail: kerscher{at}zbc.klinik.uni-frankfurt.de)
Accepted July 19, 2001
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SUMMARY |
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Key words: Alternative NADH:ubiquinone oxidoreductase, Alternative NADH dehydrogenase, Mitochondrial import, Yeast, Yarrowia lipolytica, Mitochondria
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INTRODUCTION |
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By contrast, alternative NADH:ubiquinone oxidoreductases are single subunit enzymes that contain one molecule of non-covalently bound FAD as redox cofactor and do not contribute to the proton gradient across the respiratory membrane. In eukaryotes they may reside either on the external or the internal face of the mitochondrial inner membrane. The physiological significance of this fundamental functional difference within a family of closely related enzymes is unknown. Various plants and fungi are equipped with different sets of alternative NADH:ubiquinone oxidoreductases (Kerscher, 2000). Bakers yeast, Saccharomyces cerevisiae, has three alternative NADH:ubiquinone oxidoreductases. Two of these, called SCNDE1 and SCNDE2, are external enzymes (Luttik et al., 1998; Small and McAlister-Henn, 1998), whereas SCNDI1 is an internal enzyme (Marres et al., 1991; de Vries et al., 1992). S. cerevisiae presents a special case among ascomycetous fungi, since ethanolic fermentation, rather than respiration, is the preferred mode of glucose utilization (Lagunas, 1986). In this organism, complex I is lacking (Büschges et al., 1994) and SCNDI1 is the only NADH:ubiquinone oxidoreductase present within the mitochondrial matrix. By contrast, the obligately aerobic yeast Y. lipolytica, which does possess complex I, has only one single alternative enzyme, which is encoded by the NDH2 gene and resides on the external face of the mitochondrial inner membrane (Kerscher et al., 1999).
These two organisms may illustrate key questions regarding the metabolic function of external and internal alternative NADH:ubiquinone oxidoreductases in ascomycetous fungi: Are there separate metabolic pools for NADH in the cytoplasm and in the mitochondrial matrix or do redox shuttles exist? Do these operate in both directions? Is there competition between complex I and alternative enzyme(s) for the substrates NADH and ubiquinone? What are the consequences of NADH oxidation via complex I versus via the alternative enzyme? To address such questions, we constructed strains of Y. lipolytica that possess either complex I only or complex I and an internal version of alternative NADH:ubiquinone oxidoreductase. We found that the construction of strains that lacked complex I was feasible only when an internal alternative enzyme was present.
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MATERIALS AND METHODS |
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Expression of internal YLNDH2
Plasmid pUB4 was constructed from the Y. lipolytica/E. coli shuttle vector pINA443 (Barth and Gaillardin, 1996) by removing the URA3 marker by digestion with BamHI and SalI and replacing it with the Klebsiella pneumoniae hygromycin B phosphotransferase (HygBR) gene (Gritz and Davies, 1983) under the control of a strong hybrid promoter, consisting of four direct repeats of UAS1B from Y. lipolytica XPR2 in front of the minimal promoter of Y. lipolytica LEU2 (Madzak et al., 1999; Madzak et al., 2000). The new marker gene was generated by PCR using primers BamHI/Promc2 (5'-GTGGATCCAGGCCGTTGAGCACC-3') and Prom/nc (5'-TGTGGATGTGTGTGGTTGTATG-3') to amplify the hybrid promoter out of plasmid pINA1051 and primers Hyg/c (5'-ATGAAAAAGCCTGAACTCACC-3') and SalI/Hyg/nc (5'-GTCGTCGACTATTCCTTTGCCCTC-3') to amplify the HygBR ORF out of plasmid pDHHyg, followed by blunt end ligation and reamplification using primers BamHI/Promc2 and SalI/Hyg/nc. Cloning via BamHI and SalI was followed by the removal of AatII and EcoRI sites within the HygBR ORF by site directed mutagenesis according to the QuikChange protocol (Stratagene).
Plasmid pUB22, containing the NDH2i fusion gene encoding the internal version of the Y. lipolytica alternative NADH:ubiquinone oxidoreductase under the control of the NUAM promoter, was generated in the following way: inverse PCR using primers 75kSK1 (5'-GAGACGTCGGGCGGAGAC-3') and 75kSK2 (5'-GGATAGTTCGAGGATAGTGGAG-3') on genomic clone pB7 yielded a product that was gapped for the NUAM open reading frame except for the presequence (amino acids 1-34). This was blunt-end ligated with another PCR product, encompassing a truncated version of the NDH2 open reading frame, starting at amino acid position 100, which was changed from Asn to Asp, generated using primers YLNDH2SK1 (5'-GACCCCTCCGACCAGTTGC-3') and YLNDH2SK2 (5'-AGAGATATCACGGCCGAAGAC-3') on genomic clone pE8 (Kerscher et al., 1999). In the resulting clone, called pUB5, a ClaI site was generated upstream of the NDH2i start codon at position 32 to 27 by site-directed mutagenesis, yielding clone pUB5ClaI. A 4.3 kb SalI fragment from pUB5ClaI, containing the NDH2i fusion gene flanked by 1.5 kb and 1.3 kb of upstream and downstream sequence from the NUAM locus, respectively, was subcloned into pUB4.
Expression of other alternative NADH:ubiquinone oxidoreductases
To create the YLNUAM-ECNDH fusion, genomic clone pB7 was gapped for the NUAM open reading frame except for the presequence (amino acids 1-34) by inverse PCR as described above. The product was blunt-end ligated with another PCR product entailing the complete ECNDH (Young et al., 1981) open reading frame in which the TTG initiation codon had been replaced by alanine and aspartate codons, generated on E. coli genomic DNA using primers ECNDH1 (5'-GCCGAGACTACGCCATTGAAAAAG-3') and ECNDH2 (5'-ATGCAACTTCAAACGCGGAC-3'). Subcloning of a 4.3 kb SalI fragment into pUB4 yielded plasmid pUB10. A clone with two 4.3 kb SalI inserts in tandem orientation was termed pUB11.
To express the SCNDI1 gene in Y. lipolytica, genomic clone pE8 was gapped for the entire YLNDH2 open reading frame and 355 bp of upstream sequence by inverse PCR using primers NDH2inv1 (5'-CTCATACGGGCGGTATTAC-3') and NDH2inv2 (5'-AGAGTTGCAGCTTCTCCATC-3'). The product was blunt-end ligated with another PCR product entailing the complete SCNDI1 open reading frame under the control of the same strong hybrid promoter that was also used to express the hygromycin B phosphotransferase (HygBR) marker gene of plasmid pUB4. The insert was generated by PCR using primers BamHI/Promc2 and Prom/nc to amplify the hybrid promoter out of plasmid pINA1051, and primers SCNDI1SK1 (5'-ATGCTATCGAAGAATTTGTATAG-3') and (5'-GTGTTAGGTTTTGTTTAGAGG-3') to amplify the SCNDI1 open reading frame out of plasmid pMV5 (Marres et al., 1991), followed by blunt-end ligation and reamplification using primers BamHI/Promc2 and SCNDI1SK2.
Mutagenesis of the SCNDI1 processing site, in order to convert the 1 arginine into a 3 arginine by the insertion of two alanine codons behind this arginine codon was carried out by inverse PCR using 5'-phophorylated primers SCNDImut1 (5'-AGCTCTGGTGGAAGCGAATCTGAC-3') and SCNDImut2 (5'-GCTTCCACAGGGGTGGAAAACTC-3'), followed by religation. Subcloning of a 4.5 kb SalI fragment into pUB4 yielded plasmids pUB27 (3 arginine) and pUB28 (1 arginine).
Y. lipolytica techniques
Y. lipolytica genetic techniques such as transformation and sporulation were carried out as described (Barth and Gaillardin, 1996). For the determination of growth rates, complete media containing 5 g/l yeast extract, 10 g/l bacto peptone and 10 g/l of glucose or 4 g/l of sodium acetate, adjusted to pH 5.0, were used. 50 µg/ml hygromycin B (Invitrogen) was added after sterilization. 100 ml portions of media were inoculated in 1 l Erlenmeyer flasks with baffled indentations at a density of 1x105 cells/ml with cells pre-grown in the same medium and shaken at 220 rpm. OD600 was monitored at 4 hour intervals using a Hitachi U1100 spectrophotometer.
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RESULTS |
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To assess the DQA resistance conferred by plasmid pUB22 in more quantitative terms, GB5.2, a haploid ndh2 strain, was transformed with either the empty vector pUB4 or with plasmid pUB22 (Fig. 2). Transformants were cultivated in glucose-containing complete media under hygromycin B selection to a density of approximately 1x108 cells/ml. 1 µl each of dilutions containing 10, 100 or 1000 cells/ml were plated on glucose-containing complete media with increasing concentrations of the complex I inhibitor DQA (Fig. 3). Although all strains were able to grow in the presence of up to 0.25 µM DQA, only strains that possessed the NDH2i gene could survive in the presence of DQA concentrations ranging from 0.75 µM up to 10.0 µM. However, growth was significantly delayed at higher inhibitor concentrations (data not shown). It should be noted that strains in which complex I was present displayed the convoluted colony morphology typical for mycelial growth, while the nuam
pUB22 strain produced colonies that were predominantly smooth.
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Plasmid pUB10, which harbours the YLNUAM-ECNDH fusion, conferred some resistance to DQA. However, when compared with the YLNUAM-NDH2 fusion, colony growth was slower and the survival rate of ndh2 pUB10 cells declined sharply at DQA concentrations of 1 µM or higher (Fig. 4). This may be explained by a low expression level of the E. coli enzyme, due to divergent codon usage. Consistent with this hypothesis, the YLNUAM-ECNDH fusion showed a clear gene dosage effect. Plasmid pUB11, which contains two gene copies of the YLNUAM-ECNDH fusion, resulted in markedly elevated DQA resistance (Fig. 4).
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On complete media containing acetate, the specific growth rate of the GB5.2 strain that lacked alternative NADH:ubiquinone oxidoreductase was reduced by about 20% compared with the strain that possessed the internal version of this enzyme. The specific growth rate of the nuam pUB22 strain could not be assayed since no change in OD600 could be observed over 4 days. This strain did grow after incubation for 7 days, but then growth rates varied between different batches.
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DISCUSSION |
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Here we show that a strain that carries both complex I and a genetically engineered internal version of NDH2 displays a virtually identical growth rate on glucose, and an even higher growth rate on acetate when compared with the ndh2 strain. This may indicate either that the internal alternative enzyme does not efficiently compete with complex I for the substrates NADH and ubiquinone or that the reduction in ATP yield that results from the action of the alternative enzyme is not detrimental to the cell. An internal alternative enzyme may even be beneficial in preventing over-reduction of the mitochondrial matrix.
In sharp contrast to the mild phenotype of ndh2 strains, we were unable to isolate haploid strains carrying deletions in genes for highly conserved subunits of complex I, proving that complex I is essential in Y. lipolytica. This result was surprising, since deletion of highly conserved subunits of complex I had previously been reported in other ascomycetous fungi such as N. crassa (Videira, 1998) and Aspergillus niger (Prömper et al., 1993). Survival of such mutant strains had been explained by postulating the presence of internal alternative NADH:ubiquinone oxidoreductase in these species, in analogy to S. cerevisiae, where respiratory chain complex I is absent (Büschges et al., 1994) but internal alternative NADH:ubiquinone oxidoreductase is present instead (de Vries and Grivell, 1988; Marres et al., 1991). Here we show that Y. lipolytica can survive without complex I only when NDH2 is redirected to the internal face of the mitochondrial inner membrane by the N-terminal attachment of the NUAM mitochondrial targeting signal.
This situation is remarkably different from that in S. cerevisiae, which lacks complex I and can even survive without SCNDI1 (Marres et al., 1991). scndi1 strains, which have no internal NADH:ubiquinone oxidoreductase at all, are still able to generate ATP by oxidative phosphorylation, as shown from their lack of a petite phenotype on fermentable substrates and their ability to grow on highly reduced, nonfermentable carbon sources such as ethanol (Marres et al., 1991).
The phenotype of scndi1 strains had initially been explained by claiming that with highly reduced substrates such as glucose or ethanol, NADH production in the cytoplasm and transfer of redox equivalents to the mitochondrial ubiquinone pool by external alternative NADH:ubiquinone oxidoreductase or alternative pathways is sufficient to permit ATP synthesis by oxidative phosphorylation (Marres et al., 1991). However, recent findings indicate that survival of the scndi1
mutant depends on NADH generated in the mitochondrial matrix and transferred into the cytoplasm by the ethanol-acetaldehyde shuttle (Bakker et al., 2000). Unlike most of the well-studied shuttles for the transfer of redox equivalents into the mitochondrial matrix, which involve active transport steps (Dawson, 1979), this shuttle is reversible (von Jagow and Klingenberg, 1970; Bakker et al., 2000). A double mutant strain carrying deletions in both SCNDI1 and ADH3, the gene encoding mitochondrial alcohol dehydrogenase, exhibited a reduction in biomass yield on glucose and increases in ethanol production and respiratory quotient, indicative of respirofermentative metabolism in aerobic, glucose-limited chemostat cultures. By contrast, both single deletion strains exhibited fully respiratory growth under the same conditions (Bakker et al., 2000). However, scndi1
strains cannot grow on highly oxidised, nonfermentable substrates such as acetate (Marres et al., 1991). Since it may be argued that during growth on acetate NADH produced in the citric acid cycle could be transferred into the cytoplasm via the ethanol-acetaldehyde shuttle and could then be fed into the respiratory chain, this finding clearly demonstrates that the metabolic capacity of this shuttle system is limited.
The enzymes necessary for an ethanol-acetaldehyde shuttle may be present in Y. lipolytica. Three genes for alcohol dehydrogenases (GenBank accession nos AAD51737, AAD51738 and AAD51739) have been identified in this species. Pyruvate decarboxylase is encoded in the genomes of several yeasts, including non-fermentative species. Three STS sequences (GenBank accession nos AL411453, AL412002 and AL412286) for two Y. lipolytica homologs of S. cerevisiae pyruvate decarboxylase were identified as part of a random genomic sequencing program of 13 yeast species (Casaregola et al., 2000; Souciet et al., 2000). If an ethanol-acetaldehyde shuttle does operate in Y. lipolytica, its efficiency must be lower than in S. cerevisiae. Insufficient redox shuttle activity would explain why Y. lipolytica strains that, owing to a deletion in one of the genes for the central subunits of complex I, lack all internal NADH:ubiquinone oxidoreductase activity were unable to survive even on complete media containing highly reduced carbon sources such as glucose.
The results presented here also shed new light on the mitochondrial import of NDH2. The fact that NDH2i could substitute for complex I demonstrates that the internalised enzyme does associate with the inner face of the mitochondrial inner membrane and is fully capable of interacting with the ubiquinone pool of the respiratory chain. As Y. lipolytica does not normally contain an internal alternative enzyme, this strongly suggests that membrane association does not require interaction with any protein component in the mitochondrial inner membrane. Also, integration of the enzymes redox prosthetic group, a non-covalently bound molecule of FAD, has to take place either before mitochondrial import or must be possible in a similar way both in the cytosol and in the mitochondrial matrix.
Successful redirection of NDH2 just by attaching a mitochondrial targeting signal supports the notion that the association of alternative NADH:ubiquinone oxidoreductases with the internal face of the mitochondrial inner membrane was brought about by one single evolutionary step following gene duplication, namely the acquisition of a matrix targeting sequence by an originally external alternative enzyme.
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ACKNOWLEDGMENTS |
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