The yeast Ura2 protein that catalyses the first two steps of pyrimidines biosynthesis accumulates not in the nucleus but in the cytoplasm, as shown by immunocytochemistry and Ura2-green fluorescent protein mapping.

The Ura2 multidomain protein catalyses the first two steps of pyrimidines biosynthesis in Saccharomyces cerevisiae. It consists of a 240 kDa polypeptide which contains carbamyl phosphate synthetase and aspartate transcarbamylase domains. The Ura2 protein was believed to be nucleoplasmic, since one of the aspartate transcarbamylase reaction products, monophosphate, was reported to be precipitated by lead ions inside nuclei. However, this ultracytochemical approach was recently shown to give artifactual lead polyphosphate precipitates, and the use of cerium instead of lead failed to reveal this nucleoplasmic localization. Ura2 localization has therefore been undertaken by means of three alternative approaches based on the detection of the protein itself: (a) indirect immunofluorescence of yeast protoplasts; (b) immunogold labelling of ultrathin sections of embedded yeast cells (both approaches using affinity purified primary antibodies directed against the 240 kDa Ura2 polypeptide chain, or against a 22 residue peptide specific of the carbamyl phosphate synthetase domain); and (c) direct fluorescence of cells expressing an Ura2-green fluorescent protein hybrid. All three approaches localize the bulk of Ura2 to the cytoplasm, whereas the signals associated with the nucleus, mitochondria or vacuoles are close to or at the background level.

Life-cycle-dependent changes of aspartate carbamoyltransferase localization in membranes of Saccharomyces cerevisiae--centrifugal elutriation and ultracytochemical study.

Exponential culture of a Saccharomyces cerevisiae strain with overexpressed aspartate carbamoyltransferase activity (ACTase) was chilled in ice and fractionated by centrifugal elutriation to several cell populations of increasing cell mass. The enzyme activity which belongs to the pyrimidine biosynthesis pathway, was detected in situ by a specific ultracytochemical reaction: the ACTase byproduct, monophosphate, was precipitated by cerium ions to cerium phosphate. During the outgrowth of nonbudding daughter cells (zero cells) the label appeared first in membranes of nuclear envelope and of mitochondria. In larger zero cells, this label appeared also in the endoplasmic reticulum, microvesicles and plasmalemma. In budding mother cells, the label was conspicuous in the whole cell-membrane complex. In most aged cells the ACTase activity was not detectable. The presence of ACTase activity in membranes of compartments conveying glycoproteins via the secretory pathway remains to be explained. To confirm the in situ detection of ACTase activity in membranes, we assayed the enzyme activity in both the 10,000 g sediment and supernatant prepared from yeast homogenate precentrifuged at 3000 g. From 23 to 43% of ACTase activity was detected in the sediments including membranes of wild-type and ACTase-overexpressing strains.

Carbamyl-phosphate synthetase domain of the yeast multifunctional protein Ura2 is necessary for aspartate transcarbamylase inhibition by UTP.

In Saccharomyces cerevisiae, the first two reactions of pyrimidine biosynthesis are catalyzed by the multifunctional protein Ura2 carrying both carbamyl-phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) enzyme activities. In order to study how UTP regulates both of these activities mutant strains were constructed: one strain which expressed the Ura2 protein fused to the green fluorescent protein, and two strains expressed truncated Ura2 proteins. These strains exhibited a phenotype associated with a modified regulation of the pyrimidine pathway. Results presented in this report provide arguments in favor of a single UTP binding site located on the CPSase domain, and support a model in which ATCase activity is inhibited by UTP only when it can interact with the CPSase domain.

Cerium-based ultracytochemical localization of aspartate transcarbamylase activity in the cell membrane complex of Saccharomyces cerevisiae.

Aspartate transcarbamylase (ATCase) activity was localized ultracytochemically in the yeast Saccharomyces cerevisiae by precipitation of its reaction product orthophosphate as cerium phosphate. We prefixed yeast cells with ice-cold 1% glutaraldehyde for 30 min which preserved 80% of ATCase activity. Cells were washed and incubated with ATCase substrates (aspartate, carbamyl phosphate) plus cerium chloride, and postfixed by osmium tetroxide. In cells from exponential batch cultures, deposits of cerium phosphate delineated simultaneously or alternatively membranes of the secretory pathway: nuclear envelope, endoplasmic reticulum, Golgi complex and the plasmalemma; mitochondrial membranes and intramitochondrial fibrous component were labelled as well. Deposits of cerium phosphate were never observed in the nucleoplasm. Cells incubated in the absence of cerium or ATCase substrates and mutant S. cerevisiae cells lacking ATCase activity served as controls. Small round electron-dense condensates were found to be randomly distributed within some cells, both in control and experimental runs, in the nucleoplasm, cytoplasm and mitochondrial matrix and represented undefined osmicated endogenous compounds. Our results suggest that the synthesis of pyrimidine precursors occurs in membranes, where compounds such as UDP-glucose and CDP-diglycerides are needed for membrane and/or yeast cell wall synthesis. The possible contribution of ATCase activity found in the nuclear envelope to nucleic acid synthesis remains to be clarified.

Yeast carbamoyl-phosphate-synthetase--aspartate-transcarbamylase multidomain protein is phosphorylated in vitro by cAMP-dependent protein kinase.

The first two steps of de novo pyrimidine synthesis in Saccharomyces cerevisiae are catalyzed by a multifunctional protein, coded by the URA2 gene and which has the carbamoyl-phosphate (CPSase) synthetase and aspartate transcarbamylase (ATCase) activities. The native enzyme purified from protease-B-deficient URA2-transformed cells, was phosphorylated in vitro using catalytic subunits of pure cAMP-dependent protein kinase. After electrophoresis under denaturing conditions, a single 240-kDa species was found to be phosphorylated. Trypsin digestion of this species gave a single, very acidic phosphopeptide upon isoelectric focussing. Purification by HPLC followed by amino acid sequencing of this peptide, showed a phosphoserine at the expected consensus sequence Arg-Arg-Phe-Ser. Knowledge of the URA2 gene sequence allowed the site to be located in the peptide link between dihydroorotase-like and ATCase domains. Such a location may explain why phosphorylation of the URA2 protein changed neither CPSase and ATCase activities nor their sensitivity to UTP, their common specific inhibitor.

Pyrimidine biosynthesis in Saccharomyces cerevisiae: the ura2 cluster gene, its multifunctional enzyme product, and other structural or regulatory genes involved in de novo UMP synthesis.

There are six enzymatic steps in the de novo biosynthesis of uridine monophosphate (UMP). In yeast, six structural genes (ura2, ura4, ura1, ura5, ura10, and ura3) and one regulatory gene (PPR1) are involved in this metabolic pathway. Gene ura2 codes for a multifunctional protein that carries the first two enzymatic activities of the pathway, i.e., carbamylphosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Gene ura2 has been cloned and sequenced, revealing the presence of three open reading frames, one of which codes for the multifunctional protein, a polypeptide of 2212 amino acids, with a mRNA of 7 +/- 0.3 kilobases. Expression of gene ura2 is regulated at the transcriptional level. As I indicate here, it could also be controlled at the posttranscriptional level since all the consensus sequences for a 1.2-kilobases intron are present in the coding sequence of the open reading frame. The deducted amino acid sequence has allowed the identification of four domains. Starting from the amino terminus of the protein, these are glutamine amido transferase, CPSase, a domain that resembles dihydroorotase (DHOase-like) but does not have DHOase activity, and ATCase. There are also two sites of interest that match known concensus phosphorylation sites; one is located in the distal part of the CPSase domain, the other in the connector region between DHOase-like and ATCase domains. The protein has been purified as a multienzyme aggregate and as a multifunctional protein. The latter form, when isolated from a protease B deficient strain of Saccharomyces cerevisiae, contained mostly polypeptide chains of 220 kilodaltons. Work is currently in progress to determine the site(s) of phosphorylation of this protein in vitro. ATCase activity of both wild-type and protease-deficient strains has been found to be localized in the nucleus. Channeling of carbamyl phosphate, the first intermediate in the pathway, has been demonstrated both in vitro and in permeabilized cells. The other genes of UMP biosynthesis, except for ura5, are regulated by induction of their transcription by the combined action of the product of the ppr1 gene and the inducer, dihydroorotate. Dihydroorotate dehydrogenase activity was found in the cytoplasm. Two isoenzymes of orotate phosphoribosyl transferase have been found, coded for by ura5 and ura10. The products of genes ura10 and ura3 are proposed to participate in the channeling of orotidine monophosphate. The discussion considers the problem posed by the isolation of both multienzyme complexes and multifunctional proteins resulting from the expression of the same cluster genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Proteolytically induced changes in the molecular form of the carbamyl phosphate synthetase-uracil-aspartate transcarbamylase complex coded for by the URA2 locus in Saccharomyces cerevisiae.

When a uracil-auxotrophic yeast strain is grown under uracil-limiting conditions, the aspartate transcarbamylase activity found in crude extracts shows a variation in sensitivity to feedback inhibition by uridine 5'-triphosphate. In this study we correlated this variation with changes in the molecular form of the carbamyl phosphate synthetase-uracil-aspartate transcarbamylase complex. Carbamyl phosphate synthetase-uracil (molecular weight, 240,000) and uridine 5'-triphosphate-insensitive aspartate transcarbamylase (molecular weight, 140,000) were present separately in extracts from cells collected in the early exponential phase; this was in contrast to the presence of a single high-molecular-weight form (molecular weight, about 900,000) bearing both activities in extracts from stationary-phase cells. The lack of sensitivity to uridine 5'-triphosphate by aspartate transcarbamylase was delayed by adding uridine 5'-triphosphate before cell disruption and was prevented completely by adding phenylmethylsulfonyl fluoride. Thus, this event was attributed to a transient serine protease activity detected only in early exponential-phase cell extracts. However, even in the presence of phenylmethylsulfonyl fluoride, a sucrose density gradient analysis in the absence of uridine 5'-triphosphate revealed a change in the aggregation state of the complex which might have occurred in vivo. None of these events was observed in extracts from cells that lacked protease B activity (strain HP232-2B).

Fine structure of the URA2 locus in Saccharomyces cerevisiae. II. Meiotic and mitotic mapping studies.

The URA2 locus codes for a multifunctional enzyme complex carrying aspartate transcarbamylase (ATCase) and carbamyly phosphate synthetase (CPSase) activities. Three different types of ura2 mutants were tested in meiotic and mitotic recombination experiments: ura2A mutants devoid of ATCase activity, ura2C mutants devoid of CPSase activity and ura2B mutants devoid of both activities. All the ura2A mutations were found to be clustered at one end of the URA2 locus, called zone A, while the ura2C mutations were localized in a region at the other end, called zone C. All but two ura2B mutations (most of them suppressible) were distributed throughout zone C; the two ura2B exceptions which are small deletions, mapped in zone A. On the meiotic as well as on the mitotic map an intermediary or dead-space zone is located between zones A and C. No mutation has yet been found to map in this zone. The relative lengths of the three zones A, intermediary and C are 1 :2-3 :3-4, respectively. These data are consistent with the hypothesis that the URA2 locus consisting of at least two cistrons: C (CPSase) and A (ATCase), is transcribed into a single polycistronic message in the direction C to A. However, alternative hypotheses in reference to Peterson and MacLaughlin's observations (1973) are discussed.