A cDNA encoding the dihydroorotate dehydrogenase (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.

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The cytochemical technique using the in situ precipitation of orthophosphate ions liberated specifically by the aspartate carbamoyltransferase (ATCase) (EC 2 .1 .3 .2) reaction indicated that in Saccharomyces cerevisiae this enzyme is confined to the nucleus. This observation is in accordance with the result reported by Bernhardt and Davis (1972), Proc . Nati . Acad . Sci. U. S. A. 69 :1868-1872) on Neurospora crassa . The nuclear compartmentation was also observed in a mutant strain lacking proteinase B activity . This finding indicates that this proteinase is not involved in the nuclear accumulation of ATCase, and that the activity observed in the nucleus corresponds to the multifunctional form associated with the uracil path-specific carbamoylphosphate synthetase and sensitive to feedback inhibition by UTP. In a ura2 strain transformed by nonintegrated pFL1 plasmids bearing the URA2-ATCase activity encoding gene, the lead phosphate precipitate was observed predominantly in the cytoplasm . This finding enhances the reliability of the technique used by eliminating the possibility of an artifactual displacement of an originally cytoplasmic reaction product during the preparation of the material for electron microscopy . On the other hand, nuclei isolated under hypoosmotic conditions do not exhibit the ATCase activity that is recovered in the cytosolic fractions after differential centrifugation of the lysate in Percoll gradient . A release of the protein from the nuclei during the lysis step, consistent with its nucleoplasmic localization, is postulated .Although most metabolic reactions are known and in a number of cases well-described in vitro, much less information is available concerning the conditions under which these reactions proceed in the cell . Subcellular localization of the enzymes, compartmentation, channeling, interactions with the cellular environment, relative concentration of enzymes and intermediary metabolites have to be considered (1-4) .Reported studies on the intracellular compartmentation of enzymes have centered on organelles such as vacuoles and mitochondria, whose role as "compartment" is ensured by their semipermeable membrane functioning as an effective permeability barrier between their interior and the cytosol (3-7) . As far as the nucleus is concerned, its definition as a cellular compartment is, at present, more ambiguous . A conceptual difficulty lies in the presence of the pore complex in the nuclear envelope, rendering its analogy with a true semipermeable membrane rather questionable (8, 9). Furthermore, because of the well-known leak-out of the soluble nuclear material during the procedure of cell fractionation, very little is known about the nuclear localization of enzymes other than those firmly bound to the chromatin .Enzymes catalyzing the reactions that liberate orthophos-790 phate ions can be localized through the in situ precipitation of lead phosphate, detectable by electron microscopy . In rat and mouse hepatocytes, this method was used to localize ornithin...