Purine-cytosine permease of Saccharomyces cerevisiae. Effect of external pH on nucleobase uptake and binding.
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 (Ktapp, apparent Michaelis constant and Vm) and on the binding constants of these three solutes (Kdapp, apparent half-saturation constant and Bmax, total binding sites) determined on plasma membranes. For pAB4, the variations of Ktapp and Vm were the same for the three bases, i.e. an increase in Ktapp when the pH increased and a maximum Vm around pH 5. For pAB25, Ktapp 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 Vm for adenine, and there was a continuous decrease when the pH increased in the Vm 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 phenomenon was observed. However, the curves showing the variation of Kdapp 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 microM. An amino acid displaying a pK of 3.8 was determined; this value was shifted to pK 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 (Kd of 14.4 +/- 4.3 microM) and a shift in the pK of the amino acid towards a more acidic pH (about 2.9). The pK 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.
Brèthes
D
D
Institut de Biochimie Cellulaire du Centre National de la Recherche Scientifique (ERS 0022), Bordeaux, France.
Napias
C
C
Torchut
E
E
Chevallier
J
J
D000225:Adenine / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003596:Cytosine / Q000378:metabolism*; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D033702:Nucleobase Transport Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D002352:Carrier Proteins; C043181:FCY2 protein, S cerevisiae; D007042:Hypoxanthines; D008024:Ligands; D026901:Membrane Transport Proteins; D033702:Nucleobase Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D019271:Hypoxanthine; D003596:Cytosine; D000225:Adenine