Functional Analysis of Mutated Purine-Cytosine Permease from Saccharomyces cerevisiae

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 K t (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 sitedirected 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 K t (app) of uptake were correlated with variations of the binding K d (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 pK a of an ionizable amino acid residue of the unliganded permease. These two amino acid residue changes induced a shift of the pK a 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.In the yeast Saccharomyces cerevisiae, the purine-cytosine permease (PCP) 1 mediates cotransport through the plasma membrane of proton and purine bases (adenine, hypoxanthine, and guanine) or a pyrimidine base (cytosine) (1-6). The FCY2 gene, encoding for PCP, has been cloned (7) and sequenced (8), thus enabling studies of the relationships between the deduced amino acid sequence of the permease (a polytopic protein consisting in 533 amino acid residues with a molecular mass of 58.2 kDa) and the transport mechanism.An important question for the mechanism of base transport through the plasma membrane is the characterization of the amino acid residues involved in the binding of ligands. It has been shown that the 4 bases bin...

New Plasmid System To Select for Saccharomyces cerevisiae Purine-Cytosine Permease Affinity Mutants

“…These new results and the I371, N374, I375, P376, and N377 substitutions previously studied (2,6,8) clearly show that this region is involved in maintaining the PCP in a functional configuration, probably playing an active part in the conformational changes needed for solute translocation. In particular, asparagine 377 is thought to play an essential role in the solute-binding site, which is consistent with our results, because more than 70% of the selected mutations (41 of 58) affected this residue.…”

“…Disruption of this gene abolishes active transport, despite the presence in the yeast genome of FCY21 and FCY22 (accession number X97346), which belong to the same gene family. Several mutants with modified uptake abilities have been selected in vivo (2,4,6), and their analysis has led to the generation of a functional model for this permease (7).To test this model and to investigate further the structurefunction relationship of the PCP, we isolated additional fcy2 affinity mutant alleles by various types of in vivo positive selection, making use of the broad specificity of this permease. PCP affinity mutants were isolated as previously described (4), by selecting cells able to overcome growth inhibition due to the outcompetition of two of the PCP substrates for entry into the cell.…”

“…Disruption of this gene abolishes active transport, despite the presence in the yeast genome of FCY21 and FCY22 (accession number X97346), which belong to the same gene family. Several mutants with modified uptake abilities have been selected in vivo (2,4,6), and their analysis has led to the generation of a functional model for this permease (7).…”

A Microarray-Based Genetic Screen for Yeast Chronological Aging Factors

“…LCL3 encodes a protein with homology to Staphylococcus aureus nuclease [29]. Three of the long-lived mutants were involved in either de novo purine biosynthesis ( ADE3 and ADE4 ) or purine import ( FCY2 ) [30]–[32]. Ade4 catalyzes the first step of the pathway, while Ade3 functions in one-carbon metabolism, which donates tetrahydrofolate-linked carbon units for synthesis of the purine ring (see Figure 3B).…”

The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility

“…The importance of these structural elements in solute transport and binding is supported by extensive amino acid substitution and mutational analysis in S. cerevisiae FCY2 and A. nidulans FCYB (Bréthes et al 1992;Ferreira et al 1997Ferreira et al , 1999aKrypotou et al 2012). Most of the mutations that affect cation binding, solute binding, or transport locate to TMI, III, VI and especially TMVIII that, in part, form the cation and solute binding pocket (Fig.…”