Purine–cytosine permease of Saccharomyces cerevisiae

The cloned FCY2 gene (strain pAB4) of the purine‐cytosine permease (PCP) of Saccharomyces cerevisiae and the cloned allele fcy2‐21 (strain pAB25) introduced into an S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus [Weber, E., Rodriguez, C., Chevallier, M. R. & Jund, R. (1990) Mol. Microbiol. 4 , 585–596] were studied. The influence of external pH (varying over 3.5–6) has been analysed on the uptake of adenine, hypoxanthine and cytosine ( K tapp' apparent Michaelis constant and V m ) and on the binding constants of these three solutes ( K dapp' apparent half‐saturation constant and B max' total binding sites) determined on plasma membranes. For pAB4, the variations of K tapp and V m were the same for the three bases, i.e. an increase in K tapp when the pH increased and a maximum V m around pH 5. For pAB25, K tapp values varied in the same way and were significantly higher for the three bases than those found in pAB4. There was almost no variation of V m for adenine, and there was a continuous decrease when the pH incréased in the V m of hypoxanthine and cytosine. Equilibrium binding measurements were performed for the three bases with plasma membrane isolated from pAB4 and pAB25. One single class of binding sites was detected. For pAB4, the affinity increased when the pH decreased for the three bases. The affinity of PCP for adenine was always greater than for cytosine or hypoxanthine. For pAB25, the same phenomenom was observed. However, the curves showing the variation of K dapp as a function of pH were shifted towards more acidic pH values. A model was used to fit the experimental binding data obtained with hypoxanthine for the calculation of the dissociation constants of its binding to PCP and to determine the ionization constants of an amino acid involved in ligand binding. For pAB4, at acid pH, the dissociation constant was 1.7 ± 0.4 μM. An amino acid displaying a p K of 3.8 was determined; this value was shifted to p K 4.8 when hypoxanthine was bound. For pAB25, the main effects of the mutation were a large decrease in the affinity of PCP for hypoxanthine ( K d of 14.4 ± 4.3 μM) and a shift in the p K of the amino acid towards a more acidic pH (about 2.9). The p K of this group remained similar to the value obtained with pAB4 when hypoxanthine was bound. From these data, it is proposed that the binding of hypoxanthine and H + is a random process.