Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8966-8970, October 1992 Biochemistry

Divergent evolution of pyrimidine biosynthesis between anaerobic and aerobic yeasts MARIE NAGY*, FRANCOIs LACROUTEt, AND DOMINIQUE THOMAS*: *Laboratoire d'Enzymologie and tCentre de Gdndtique Moldculaire, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France

Communicated by Boris Magasanik, July 6, 1992

A cDNA encoding the dihydroorotate dehyABSTRACT drogenase (DHOdehase; EC 1.3.3.1) of the yeast Schizosaccharomyces pombe was isolated by functional complementation in Saccharomyces cerevisiae. A divergent subcellular compartmentation of the DHOdehase of each yeast was shown. The DHOdehase from Sch. pombe was localized in the mitochondria whereas its homolog from S. cerevisiae was found to be cytosolic. The heterologous expression of the Sch. pombe enzyme in S. cerevisiae allowed us to demonstrate that the Sch. pombe DHOdehase activity requires the integrity of the mitochondrial electron transport chain. Indeed, the presence of a mutation inactivating cytochrome b abolished the complementation of a S. cerevisiae ural mutant by the corresponding Sch. pombe gene. By contrast, in vitro studies have revealed that the DHOdehase of S. cerevisiae uses fumarate as terminal electron acceptor. These results are discussed in relation to the anaerobic growth competence of the two yeasts and to the fermentative processes they use.

approach not only enables isolation of new genes but also facilitates investigations on the molecular mechanisms involved in basal metabolism, owing to the powerful molecular methods developed to study S. cerevisiae. We report here the sequence of a cDNA§ encoding the DHOdehase from Sch. pombe and the mitochondrial localization of its product when this cDNA was expressed either in Sch. pombe or in S. cerevisiae. In contrast, the S. cerevisiae DHOdehase remained in the cytosol in either yeast in which its gene was expressed. We present evidence suggesting that this divergent localization is related to the differences existing between the anaerobic growth competence of each yeast. We found that the Sch. pombe DHOdehase activity is dependent on the integrity of the respiratory chain. In addition, we determined that the S. cerevisiae DHOdehase certainly uses fumarate as terminal electron acceptor.

The conversion of dihydroorotate to orotate, catalyzed by dihydroorotate dehydrogenase (DHOdehase; EC 1.3.3.1), is the single redox reaction in the biosynthesis of pyrimidine nucleotides. DHOdehases have been extensively studied and purified from various organisms, including prokaryotes, lower eukaryotes, and mammals (ref. 1 and references therein). Emerging from these studies is the puzzling observation that considerable divergences occur in the subcellular compartmentation of this enzyme. Indeed, prokaryotic DHOdehases were found to be either associated with the membranes, as in Escherichia coli (2), or soluble, as in Lactobacillus bulgaricus (3). Eukaryotic DHOdehases are localized either in mitochondrial membranes, as in Neurospora crassa (4) and in mammals (1), or in the cytoplasm, as in Trypanosonoma brucei (5). The functional relationship between the subcellular compartmentation and the mechanism of orotate synthesis was deciphered in the case of prokaryotic membrane-associated DHOdehase. In E. coli, the localization of the DHOdehase allows it to transfer the reducing equivalents generated by dihydroorotate oxidation to the membrane-bound electron transport chain, either aerobically or anaerobically (6). In addition, from in vitro experiments, the mammalian membrane-associated DHOdehase appears to be linked to the mitochondrial electron transport system (1). The study we present here was performed to obtain further insight into the mechanisms of dihydroorotate oxidation in two lower eukaryotes, a facultatively anaerobic yeast, Saccharomyces cerevisiae, and an aerobic yeast, Schizosaccharomyces pombe. The gene encoding the Sch. pombe DHOdehase was isolated by functional complementation of a DHOdehase-deficient S. cerevisiae mutant. The general approach of cloning genes from heterologous species by functional complementation has been widely used (7, 8). This

Yeast Strains. A ural deletion derivative of the S. cerevisiae wild-type strain FL100 was used to clone the cDNA encoding the Sch. pombe DHOdehase. The S. cerevisiae isogenic strains DM277 (MATa, ade2, leu2, his3, trpl, ural::HIS3, [rho+, mit-]) and DM304 (MATa, ade2, leu2, his3, trpl, ural::HIS3, [rho', mit+]) are derived from the W303-1A strain. The mitochondrial mit- mutation present in the DM277 strain is the w7 mutation that leads to a deficient cytochrome b owing to a replacement of Glycine-131 by a serine residue (9, 10). The Sch. pombe strains were the wild type, 972h-, and its DHOdehase-deficient isogenic derivative ura3-34 (11). S. cerevisiae and Sch. pombe growth conditions (12) and genetic manipulation and yeast transformations (13) were as previously described. Plasmids. To express the S. cerevisiae DHOdehase in Sch. pombe, we used the pRL11 plasmid (14). To construct a null mutation of the S. cerevisiae URAI gene, we first inserted the HIS3 gene at the single EcoRV site of the URAI gene borne by the pFL392 plasmid (15). The resulting plasmid was digested with the restriction enzymes Cla I and BamHI, resulting in the excision of a 2.5-kilobase-pair (kbp) fragment. This fragment was used to transform the URAI, his3 strains and HIS+ transformants were selected in the presence of uracil. The pFLRec2 plasmid, expressing the Sch. pombe DHOdehase, was constructed by replacing the URA3 gene present on the pFLRecl plasmid by the TRPI gene. To sequence the cDNA encoding the Sch. pombe DHOdehase, the Not I-Not I fragment of pFLRecl was subcloned in both orientations into the bacteriophage M13mpl8, and systematic deletion subclones were generated (16). Corresponding single-stranded phage DNAs were sequenced with a Pharmacia T7 sequencing kit. Sequence analyses were performed with the Bisance service (17).

MATERIALS AND METHODS

Abbreviation: DHOdehase, dihydroorotate dehydrogenase. tTo whom reprint requests should be addressed. §The sequence reported in this paper has been deposited in the GenBank data base (accession no. X65114).

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Subcellular Fractionation and Enzyme Assays. Spheroplast formation, disruption, and fractionation were performed as described (18), except that Sch. pombe and S. cerevisiae cell walls were digested respectively by Novozym 234 (Novobiolabs, Bagsvaerd, Denmark) and Zymolyase 60,000 (Seikagaku Kogyo, Tokyo) enzymes in the presence of 1.35 M sorbitol. For subcellular fractionation experiments, DHOdehase activity was assayed by using 2,6-dichloroindophenol as electron acceptor and by following the loss of absorbance at 600 nm (1). An alternative method was used for the study of the S. cerevisiae DHOdehase that directly monitored the formation of orotate by its absorbance at 290 nm (E290 = 6.4 x 103 M-1cm-l). S. cerevisiae cell extracts were made as described (12) in the presence of 2 mM phenylmethylsulfonyl fluoride and 1 mM EDTA. After centrifugation for 30 min at 10,000 x g, the supernatant was filtered on a G-25 Sephadex column. A standard assay mixture (1 ml) contained 40 mM sodium acetate buffer (pH 6.0), cell extract containing 3-50 pug of protein, and 0.02-2 mM dihydroorotate. Three different protein concentrations were assayed for each cell extract. In each assay, the enzymatic reaction was initiated by addition of dihydroorotate to a thermally equilibrated solution. All reactions were performed at 30CC. Protein concentrations were estimated by the method of Lowry et al. (19).

RESULTS Isolation of a cDNA Encoding the Sch. pombe DHOdehase. A functional cDNA encoding the DHOdehase of Sch. pombe was cloned by complementation of the uracil auxotrophy of a S. cerevisiae ural null mutant. A Sch. pombe cDNA library was made in the pFL61 vector, which permits expression of the cDNA inserts under the control of the yeast PGK promoter (20). Two independent Ural clones were isolated from this library. By restriction mapping, these clones were found to harbor identical plasmids containing a 1.6-kbp insert. One of these plasmids was retained and called pFLRecl. The 1613-nucleotide sequence of the Sch. pombe DHOdehase cDNA encodes a 443-amino acid protein (Mr 48,000) as deduced from the longest open reading frame of the cloned cDNA (Fig. 1). A comparison of the predicted amino acid sequence with the known sequences of the DHOdehases from E. coli (21), S. cerevisiae (15), and Drosophila melanogaster (J. Rawls, personal communication) was made by using the MACAW program (22). Optimized alignment reveals that the Sch. pombe enzyme is more similar to its E. coli and D. melanogaster homologs than to the S. cerevisiae DHOdehase (Fig. 2). Indeed, among the 443 amino acid residues of the Sch. pombe DHOdehase, 136 are identical to residues of the D. melanogaster enzyme whereas only 52 are identical to residues of the S. cerevisiae enzyme. In addition, the S. cerevisiae enzyme appears to be the smallest DHOdehase known to date. Especially, it lacks an N-terminal domain carried by its eukaryotic homologs (Fig. 2). Interestingly, the calculated isoelectric points of the Sch. pombe (pI 9.7) and D. melanogaster (pI 9.5) DHOdehases are much more basic than that of the S. cerevisiae enzyme (pI 6.2). Subcellular Compartmentation of the Yeast DHOdehases. The URAI gene, encoding the S. cerevisiae DHOdehase, was previously isolated (14). This allowed us to determine the subcellular localization of the DHOdehase from each yeast species in both homologous and heterologous environments. Therefore we measured the DHOdehase activities in the mitochondrial and cytosolic fractions of the two wild-type yeasts, as well as those of the two DHOdehase-deficient yeasts, after transformation with the heterologous DHOdehase-encoding genes (Table 1). In the homologous environment, almost all S. cerevisiae DHOdehase activity was found in the cytosolic fraction whereas at least 80% of the Sch. pombe DHOdehase activity was present in the mito-

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chondrial fraction. Furthermore, both the S. cerevisiae and Sch. pombe DHOdehases, when expressed in the heterologous host, were localized in the same compartment they would reach in their original environment: the S. cerevisiae DHOdehase remained in the Sch. pombe cytosol whereas the Sch. pombe enzyme was targeted into the S. cerevisiae mitochondria. In agreement with this divergence in the subcellular localization of the two DHOdehases, the sequence of the Sch. pombe enzyme exhibits in its N-terminal part the typical features of a mitochondrial targeting signal (23): it is especially rich in basic amino acids as well as in serines and threonines and contains only a few acidic residues. As mentioned above, this domain is absent from the S. cerevisiae DHOdehase. Sch. pombe D1l0dehase Is Coupled to the Respiratory Chain. In the course of our investigations on the subcellular localization of the yeast DHOdehases, we observed that the enzymatic activity of the Sch. pombe enzyme was enhanced by addition of ubiquinone Q6 to the assay mixture, especially at low protein concentrations. By contrast, this effect was never observed with the S. cerevisiae DHOdehase. Quinones are usually considered to function in respiratory chains as links between the dehydrogenases and the next component in the sequence of the electron transport system. The mitochondrial localization of Sch. pombe DHOdehase and its ubiquinone-enhanced enzymatic activity strongly suggest the existence of a functional link between this dehydrogenase and the respiratory chain. One consequence of such a hypothesis is that complementation of a S. cerevisiae DHOdehase-deficient mutant by the Sch. pombe gene should be restricted to aerobic growth conditions. Indeed, the DM304 strain transformed by the pFLRec2 plasmid stopped dividing after only two to three generations when grown under anaerobic conditions in a medium lacking uracil (Fig. 3A). In mitochondria of S. cerevisiae, cytochrome b is the protein component of the respiratory chain that oxidizes

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Proc. Natl. Acad. Sci. USA 89 (1992)

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