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. ...

8-Azidoadenine was used as a photoaffinity reagent to characterize the purine -cytosine permease of Saccharomyces cerevisiae. It is a potent competitive inhibitor of cytosine uptake and irradiation of the cells incubated with the label induced the irreversible inactivation of cytosine uptake. Addition of excess cytosine prevented this labelling which was restricted to the outer face of the plasma membrane since it was not accumulated by the cells.In the strain with the amplified purine-cytosine permease gene the maximum cytosine uptake rate was increased 4 -5-fold relative to wild type without a modification of the Michaelis constant of uptake ( K J ; no uptake could be measured in the deleted strain. The relative amounts of specific labelling determined for the cells and for membrane preparations were 0, 1 and 4 for the null, the wild-type and the amplified strains, respectively.One major band specifically labelled by [3H]azidoadenine, corresponding to a polypeptide with an apparent molecular mass of 45 kDa, was observed in the wild type, amplified in the strain carrying the multicopy plasmid and not detected in the deleted strain. Therefore this polypeptide corresponds to the purine -cytosine permease.In the yeast Saccharomyces cerevisiae, at least two transport systems are involved in the uptake of purine and pyrimidine bases: one for uracil [l] and the other for purine bases (adenine, guanine and hypoxanthine) and a pyrimidine base (cytosine) [2 -41. This purine -cytosine permease is believed to work as a proton symporter and cation antiporter through the dissipation of an electrochemical gradient of protons built up by the H+-ATPase located in the membrane [5, 61. Study of this permease mechanism is now facilitated by recent data on the nucleotide sequence of the gene, the FCY2 gene [7], and by the opportunity to increase the amount of this protein in the yeast cell using a cloned permease gene on a multicopy plasmid [7, 81. As a first step in the study of the purine-cytosine permease mechanism, we describe conditions allowing covalent specific photoaffinity labelling of a protein involved in cytosine uptake by using 8-azidoadenine, a derivative of one of the transporter ligands [9]. Labelling was performed either in vivo (on cells) or in vitro (on plasma membrane) on different S. cerevisiae strains.After kinetic analysis of the action of the photoaffinity label on cytosine transport, we have studied the correlation between the specific covalent labelling of the cells and the inactivation of transport activity. We then measured the amount of polypeptide specifically labelled and involved in the uptake directly on cells and on membrane preparations