In vivo and in vitro studies of the purine‐cytosine permease of Saccharomyces cerevisiae

Abstract

The FCY2 gene of the purine-cytosine permease (PCP) of Saccharomyces cerevisiae and the allele fcy2-21 have been cloned on the yeast multicopy plasmid pJDB207. The corresponding plasmids were introduced into a S. cerevisiur strain carrying a chromosomal deletion at the FCY2 locus. The resulting strains were designated pAB4 and pAB25 respectively. The pAB25 strain, which carries the fcy2-21 allele, contains four amino acid changes in the open reading frame of the PCP (Weber et al., 1989). The influence of these mutations was studied on cells by determination of the uptake constants of purine bases and cytosine [apparent Michaelis constant of transport (Kldpp) and V,,,] and on plasmamembrane preparations, by measurements of binding parameters at equilibrium [(& was based on a large increase in Klap, for all ligands except adenine. Plasma membranes of each strain displayed one class of specific binding sites, Variations in Kd of 0.4 -1 pM were observed for pAB4. These slight variations had no effect on the K,.,, of uptake measured for the corresponding solutes. In contrast, using pAB25 membranes, Kd increased dramatically; 2.6 pM, 40 pM and 96 pM for adenine, cytosine and hypoxanthine, respectively. These increments were correlated to variations in KlaPp of the uptake for cytosine and hypoxanthine.Therefore, we conclude that modification in the Ktopp of uptake in the strain carrying fcy2-21 allele is merely due to a modification of the binding ability of the permease for its ligands.The translocation of a solute across biological membranes by a carrier is assumed to occur in at least four distinct steps; (a) the binding of the solute to the transporter at sites on one side of the membrane, (b) a conformational change of the solute-transporter complex, leading to the exposure of the binding sites to the other side of the membrane, (c) the dissociation of the complex releasing the solute and finally (d) a second conformational change of the unloaded carrier which restores the original orientation and conformation of the system for the catalytic cycle. When the translocation is simultaneously coupled with a cation electrochemical potential difference, so that secondary active transport can be achieved with regard to the solute, additional steps should be considered, i.e. cation binding to the transporter and/or the solute and its (EC 3.5.4.1).release. Kinetic analysis of the solute uptake and determination of the binding ability of the carrier are the first steps towards obtain insight into the mechanism of solute translocation. This approach is revealing when comparative studies are performed with mutants displaying modifications in transport function.In the yeast, Saccharomyces cerevisiae, the purine-cytosine permease (PCP) mediates the cotransport through the plasma membrane of protons and of purine bases (adenine, hypoxanthine and guanine) or a pyrimidine base (cytosine) [I -61. The utilisation of the transmembrane electrochemical potential difference in protons leads to accumulation of bases in the cytoplasm. ...

Characterization of the Saccharomyces cerevisiaeCytosine Transporter Using Energizable Plasma Membrane Vesicles

“…Activity Measurements on CL-PMV-Ligand equilibrium binding measurements were performed at 4°C on CL-PMV by a centrifugation technique as already described (26) in K ϩ buffer, pH 5.7, containing NaCl (100 mM) and [2-14 C]cytosine (620 MBq⅐mmol…”

Functional Analysis of Mutated Purine-Cytosine Permease from Saccharomyces cerevisiae

“…It has been shown that the 4 bases bind to the same site with very similar affinities and that their uptake occurs with equivalent efficencies (3,9). To identify the binding site, an interesting approach involves the isolation of mutant strains with altered affinity constants for the various ligands, as already described in the case of lactose permease of Escherichia coli (10 -12).…”

“…All these mutants displayed modifications of the uptake parameters for the different bases. We have already extensively described the triple mutant strain and shown that modifications of equilibrium binding parameters were responsible for the uptake phenotype (9).…”

Deletion of the Uracil Permease Gene Confers Cross-Resistance to 5-Fluorouracil and Azoles in Candida lusitaniae and Highlights Antagonistic Interaction between Fluorinated Nucleotides and Fluconazole

“…[ 14 C]uracil uptake transport was abolished in the fur4⌬ mutant but not in the dal4⌬ mutant, indicating that Fur4p specifically transported uracil. In wild-type strains of S. cerevisiae, the kinetic parameters of the transport of cytosine by Fcy2p and uracil by Fur4p are very similar, with Kt app values around 2 M and V max values in the range of 8 to 10 nmol/min/10 7 cells (16,28). In the C. lusitaniae wild-type strain, Fcy2p shows a greater affinity for cytosine [Kt app , 6 M] (8) than Fur4p for uracil [Kt app , 20 to 24 M], which itself has 10 times less affinity for uracil than Fur4p of S. cerevisiae (2.5 M) (16).…”