title: Functional analysis of mutated purine-cytosine permease from Saccharomyces cerevisiae. A possible role of the hydrophilic segment 371-377 in the active carrier conformation.
abstract: The purine-cytosine permease (PCP) is an active transporter located in the plasma membrane of the yeast Saccharomyces cerevisiae. This protein mediates purine (adenine, guanine, and hypoxanthine) and cytosine accumulation in the cell by using an electrochemical potential difference in proton as the energy source. Various mutant strains, with altered Kt(app) (apparent Michaelis constant of transport) of uptake for one or several bases, have already been selected. Their cloning and sequencing revealed that three of them presented substitutions in the same region of the putative sequence of the PCP: this region might correspond to the hydrophilic segment 371-377 (I-A-N-N-I-P-N). Two mutants displayed single mutations, resulting in only one amino acid residue change (N377I and N374I, respectively), and the other displayed three amino acid substitutions (I371V, I375V, and N377G). Therefore, to analyze the contribution of individual amino acid changes to the phenotype of the complex mutant, single (N377G) and double (I371V,I375V) mutants were constructed by site-directed mutagenesis. The influence of single mutations in this region was studied by measuring, for adenine, hypoxanthine, and cytosine, the uptake constants on cells and equilibrium binding parameters on plasma membrane-enriched fractions. Uptake and binding constant determinations showed that all the variations observed for the Kt(app) of uptake were correlated with variations of the binding Kd(app) for the corresponding solutes. Thus, our results emphasize the role of the two asparagine residues, located at positions 374 and 377, respectively, in the binding of the bases. In addition, the sole substitution of the 377 asparagine residue by glycine is responsible for the phenotype of the triple mutant. The effect of pH on the apparent hypoxanthine binding dissociation constant showed that the effects of N377G and N377I changes were, at least partially, due to a shift of the pKa of an ionizable amino acid residue of the unliganded permease. These two amino acid residue changes induced a shift of the pKa of this group in the unliganded, deprotonated permease about two units toward acidic pH. This result suggests that the 371-377 segment might play a key role in the proper three-dimensional structure of the active purine-cytosine permease.
authors.affiliation: Institut de Biochimie et Génétique Cellulaires du Centre National de la Recherche Scientifique, UPR 9026, 1 rue Camille Saint-Saëns, F-33077 Bordeaux Cedex, France.
authors.forename: T
authors.initials: T
authors.lastname: Ferreira
authors.forename: D
authors.initials: D
authors.lastname: Brèthes
authors.forename: B
authors.initials: B
authors.lastname: Pinson
authors.forename: C
authors.initials: C
authors.lastname: Napias
authors.forename: J
authors.initials: J
authors.lastname: Chevallier
mesh_terms: D000483:Alleles; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D019271:Hypoxanthine / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D033702:Nucleobase Transport Proteins; D017354:Point Mutation; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; C043181:FCY2 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033702:Nucleobase Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D019271:Hypoxanthine