Richard Jund

The URH1 uridine ribohydrolase of Saccharomyces cerevisiae

Abstract. In the yeast Saccharomyces cerevisiae, uridine ribohydrolase activity is important for recycling, via the salvage pathway, pyrimidine deoxy- and ribonucleosides into uracil required for the growth of strains lacking the de novo pyrimidine synthesis pathway. We have shown that not only uridine and cytidine, but also 5-fluorouridine, 5-fluorocytidine and deoxycytidine are substrates for this enzyme. We identified, cloned and characterized the corresponding URH1 gene and its physiological function was determined by the measurement of metabolic fluxes in several mutants impaired in the pyrimidine salvage pathway. Sequence comparison revealed strong homology between Urh1p and the inosine/uridine-preferring nucleosidase and inosine/adenosine/guanosine nucleoside hydrolase proteins from the parasitic organisms Crithidia fasciculata and Trypanosoma brucei brucei. Moreover, the Asp and His residues in the putative active site were conserved. Site-directed mutagenesis demonstrated that the conserved His residue is involved in catalysis. These results allow us to speculate that the structure and catalytic mechanism of Urh1p are similar to the inosine/uridine nucleoside hydrolase from C. fasciculata.

Characterization of the Saccharomyces cerevisiae FCY1 gene encoding cytosine deaminase and its homologue FCA1 of Candida albicans

By functional complementation of a fcy1 null mutant of Saccharomyces cerevisiae, we have cloned and characterized the FCY1 gene, encoding cytosine deaminase in Saccharomyces cerevisiae, and its homologue FCA1, encoding cytosine deaminase in Candida albicans. Disruption of FCY1 resulted in high resistance to 5-fluorocytosine (10−2 M) and in total loss of cytosine deaminase activity. By contrast the transformation by FCY1 or FCA1 of the haploid FCY1-disrupted host strain restored sensitivity to 5-fluorocytosine and allowed growth on cytosine, as a source of pyrimidine, or ammonium. FCA1 as opposed to FCY1 contains an intron. FCA1 and FCY1 encode respectively 150- and 158- residue proteins of 60% identity. Both Fcy1p and Fca1p share common motifs with cytidine and CMP deaminases, but homology with cytosine deaminase of E. coli could not be detected.

New insights into the pyrimidine salvage pathway of Saccharomyces cerevisiae : requirement of six genes for cytidine metabolism

Abstract Cytidine metabolism in the yeast Saccharomyces cerevisiae was analyzed by genetic and biochemical approaches. Disruption of a unique ORF (Genbank accession No. U 20865) bearing homology with eucaryotic or bacterial cytidine deaminases abolished cytidine deaminase activity and resulted in 5-fluorocytidine resistance. The gene encoding cytidine deaminase will be referred to as CDD1 (Genbank accession number AF080089). The ability to isolate mutants resistant to 5-fluorocytidine which mapped to five other loci demonstrated the existence of a complex cytidine metabolic network. Deciphering this network revealed several original features:(1) cytidine entry is mediated by the purine-cytosine transporter (Fcy2p),(2) cytidine is cleaved into cytosine by the uridine nucleosidase (Urh1p),(3) cytidine is phosphorylated into CMP by the uridine kinase (Urk1p),(4) a block in cytosine deaminase (Fcy1p), but not in cytidine deaminase (Cdd1p), constitutes a limiting step in cytidine utilisation as a UMP precursor.

Regulation of the pyrimidine salvage pathway by the FUR1 gene product of Saccharomyces cerevisiae

SummaryIn Saccharomyces cerevisiae, the protein encoded by the FUR1 gene is absolutely required for the expression of uracil phosphoribosyl transferase activity. The occurrence of semi-dominant mutations for 5-fluorouracil-(5FU)-resistance at this locus led us to clone and sequence the semi-dominant fur 1–5 allele. A single point mutation, resulting in the substitution of arginine 134 for serine, is responsible for this mutant phenotype. The fur 1–5 allele is transcribed and expressed at the same level as the wild-type allele. But, in contrast with the wild-type, the UPR Tase activity of the fur 1–5 mutant strain is stimulated in vitro by UTP and does not, therefore, correspond to a loss of feedback of UPR Tase activity. We found that uracil, as a free base, induces a significative increase in transcription and UPR Tase activity in a wild-type strain as well as in uracil-overproducing mutants which principally explains the high efficiency of the pyrimidine salvage pathway in S. cerevisiae.

Evolutionary relationship and secondary structure predictions in four transport proteins ofSaccharomyces cerevisiae

SummaryThe comparison of the amino acid sequences of four yeast transport proteins indicates that there is a questionable relatedness between the uracil permease (FUR4) and the purine-cytosine permease (FCY2), whereas the arginine permease (CAN1) and the histidine permease (HIP1) clearly originated from a common molecular ancestor. The analysis of the primary structure of these transport proteins by two methods of secondary structure predictions suggests the presence of 9–12 membrane-spanning α-helices in each polypeptide chain. These results are concordant in that 90% of the α-helices were determined by both methods to be at the same positions. In the aligned sequencesHIP1 andCAN1, the postulated membrane-spanning α-helices often start at corresponding sites, even though the overall sequence similarity of the two proteins is only 30%. In the aligned DNA coding sequences ofCAN1 andHIP1, synonymous nucleotide substitutions occur with very similar frequencies in regions where the replacement substitution (changing the amino acids) frequencies are widely different. Moreover, our data suggest that the replacement substitutions can be considered as neutral in the N-terminal segment, whereas the other regions are subject to a conservative selective pressure because, if compared to a random drift, the replacement substitutions are underrepresented.

Chromosomal mapping of the uracil permease gene of Saccharomyces cerevisiae

SummaryThe gene FUR4, coding for the uracil permease in Saccharomyces cerevisiae, was mapped on chromosome II, at a distance of 7.8 cM from the centromere on the right arm of the chromosome. In a first step, we used the chromosome loss mapping method developed by Falco and Botstein (1983) to determine on which chromosome the gene mapped. After the observation that FUR4 was closely linked to GAL10, one of the three genes forming the gal cluster (Bassel and Mortimer 1971), we could determine precisely the position of the gene on chromosome II.

In vivo mutational analysis of highly conserved amino acid residues of the small subunit Cpa1p of the carbamylphosphate synthetase of Saccharomyces cerevisiae.

The role of selected amino acid residues located in the putative catalytic domain and of two conserved histidine residues within the small subunit of the carbamylphosphate synthetase (CPS) specific to the arginine biosynthesis pathway of the yeast Saccharomyces cerevisiae was studied using site‐directed mutagenesis to change all residues to aspartic acid. Carbamylphosphate synthesis catalysed by modified CPS was tested in vivo. The C264D, H307D and H349D mutants were unable to grow on minimal medium, indicating the importance of these three residues for efficient CPS activity, whereas, four other mutated residues located in the catalytic site (including a proline residue) do not affect the growth rate. These results in comparison to those obtained with the CPS of Escherichia coli, implicate residues Cys 264 and His 349 in the glutaminase catalytic activity, and His 307 in the binding of glutamine to the active site. Using these three defective mutants, we investigated the in vivo utilization of ammonia by CPS. C264D and H307D mutants are able to use ammonia as a substrate when provided in sufficiently high concentrations (up to 200 mm). The H349D mutant, however, did not grow even at ammonium sulfate concentrations above 400 mm, suggesting that this substitution is critical to NH3‐dependent CPS activity although the ammonia binding site is presumably located within the large subunit of the enzyme.