Screening of an intragenic second-site suppressor of purine-cytosine permease from Saccharomyces cerevisiae - Possible role of Ser272 in the base translocation process

The purine-cytosine pemease from Saccharomyces cerevisiae mediates the acti ve transport through the plasma membrane of adenine, hypoxanthine, guanine and cytosine using the proton electrochemical potential difference as an en ergy source. Analysis of the activity of strains mutated in a hydrophilic s egment (371-377) of the polypeptidic chain has shown the involvement of thi s segment in the maintenance of the active three-dimensional structure of t he carrier In an attempt to identify permease domains that could interact f unctionally and/or physically with this segment, we looked for second-site mutations that could suppress the effects of amino acid changes in this reg ion, This paper describes a positive screen that has allowed the isolation of one suppressor from a permease mutant displaying the N374I change (fcy2- 20 allele), a substitution that induces a dramatic decrease in the affinity of the carrier for adenine, cytosine and hypoxanthine. The second-site mut ation corresponds to the replacement of the Ser272 residue by Leu. Its supp ressive effect is shown to be a partial restoration of the binding of cytos ine and hypoxanthine to the permease. To test whether this second-site muta tion is specific for the fcy2-20 allele, two double mutants were constructe d (Fcy2(T213I. S272L) and Fcy2p(S272L. N377G)). Results obtained with these two double mutants showed that the suppressive effect of S272 L replacemen t was not specific for the original N374I change. To understand the general effect of this amino acid replacement for the three distinct double mutant s, a strain overexpressing Fcy2p(S272L) was constructed. Kinetic analysis o f this strain showed that, by itself, the S272 L change induced an improvem ent in the base-binding step that could account for its global suppressive effect. Moreover. S272 L induced a decrease in the turnover of the permease , thus showing the involvement of S272 in the translocation process. Taking into account the topological model of the permease proposed here. this Ser residue is probably located in a transmembrane amphipathic or-helix (TM5). The location and the observed decrease in the turnover of the carrier obse rved with the S272 L change lend us to propose that S272 could be part of a hydrophilic pore involved in the translocation of the base and/or the prot on.