The deep-sea tube worm Riftia pachyptila (Vestimentifera) from hydrothermal vents lives in an intimate symbiosis with a sulfur-oxidizing bacterium. That involves specific interactions and obligatory metabolic exchanges between the two organisms. In this work, we analyzed the contribution of the two partners to the biosynthesis of pyrimidine nucleotides through both the "de novo" and "salvage" pathways. The first three enzymes of the de novo pathway, carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase, were present only in the trophosome, the symbiont-containing tissue. The study of these enzymes in terms of their catalytic and regulatory properties in both the trophosome and the isolated symbiotic bacteria provided a clear indication of the microbial origin of these enzymes. In contrast, the succeeding enzymes of this de novo pathway, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase, were present in all body parts of the worm. This finding indicates that the animal is fully dependent on the symbiont for the de novo biosynthesis of pyrimidines. In addition, it suggests that the synthesis of pyrimidines in other tissues is possible from the intermediary metabolites provided by the trophosomal tissue and from nucleic acid degradation products since the enzymes of the salvage pathway appear to be present in all tissues of the worm. Analysis of these salvage pathway enzymes in the trophosome strongly suggested that these enzymes belong to the worm. In accordance with this conclusion, none of these enzyme activities was found in the isolated bacteria. The enzymes involved in the production of the precursors of carbamyl phosphate and nitrogen assimilation, glutamine synthetase and nitrate reductase, were also investigated, and it appears that these two enzymes are present in the bacteria.

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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...