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...

The topology of subunit i, a component of the yeast F(o)F(1)-ATP synthase, was determined by the use of cysteine-substituted mutants. The N(in)-C(out) orientation of this intrinsic subunit was confirmed by chemical modification of unique cysteine residues with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. Near-neighbor relationships between subunit i and subunits 6, f, g, and d were demonstrated by cross-link formation following sulfhydryl oxidation or reaction with homobifunctional and heterobifunctional reagents. Our data suggest interactions between the unique membrane-spanning segment of subunit i and the first transmembranous alpha-helix of subunit 6 and a stoichiometry of 1 subunit i per complex. Cross-linked products between mutant subunits i and proteins loosely bound to the F(o)F(1)-ATP synthase suggest that subunit i is located at the periphery of the enzyme and interacts with proteins of the inner mitochondrial membrane that are not involved in the structure of the yeast ATP synthase.

The FCY2 gene of the purine-cytosine permease (PCP) of Saccharomyces cerevisiae and the allele fcy2-21 have been cloned on the yeast multicopy plasmid pJDB207. The corresponding plasmids were introduced into a S. cerevisiur strain carrying a chromosomal deletion at the FCY2 locus. The resulting strains were designated pAB4 and pAB25 respectively. The pAB25 strain, which carries the fcy2-21 allele, contains four amino acid changes in the open reading frame of the PCP (Weber et al., 1989). The influence of these mutations was studied on cells by determination of the uptake constants of purine bases and cytosine [apparent Michaelis constant of transport (Kldpp) and V,,,] and on plasmamembrane preparations, by measurements of binding parameters at equilibrium [(& was based on a large increase in Klap, for all ligands except adenine. Plasma membranes of each strain displayed one class of specific binding sites, Variations in Kd of 0.4 -1 pM were observed for pAB4. These slight variations had no effect on the K,.,, of uptake measured for the corresponding solutes. In contrast, using pAB25 membranes, Kd increased dramatically; 2.6 pM, 40 pM and 96 pM for adenine, cytosine and hypoxanthine, respectively. These increments were correlated to variations in KlaPp of the uptake for cytosine and hypoxanthine.Therefore, we conclude that modification in the Ktopp of uptake in the strain carrying fcy2-21 allele is merely due to a modification of the binding ability of the permease for its ligands.The translocation of a solute across biological membranes by a carrier is assumed to occur in at least four distinct steps; (a) the binding of the solute to the transporter at sites on one side of the membrane, (b) a conformational change of the solute-transporter complex, leading to the exposure of the binding sites to the other side of the membrane, (c) the dissociation of the complex releasing the solute and finally (d) a second conformational change of the unloaded carrier which restores the original orientation and conformation of the system for the catalytic cycle. When the translocation is simultaneously coupled with a cation electrochemical potential difference, so that secondary active transport can be achieved with regard to the solute, additional steps should be considered, i.e. cation binding to the transporter and/or the solute and its (EC 3.5.4.1).release. Kinetic analysis of the solute uptake and determination of the binding ability of the carrier are the first steps towards obtain insight into the mechanism of solute translocation. This approach is revealing when comparative studies are performed with mutants displaying modifications in transport function.In the yeast, Saccharomyces cerevisiae, the purine-cytosine permease (PCP) mediates the cotransport through the plasma membrane of protons and of purine bases (adenine, hypoxanthine and guanine) or a pyrimidine base (cytosine) [I -61. The utilisation of the transmembrane electrochemical potential difference in protons leads to accumulation of bases in the cytoplasm. ...

It is possible to obtain from yeast mitochondria a proteolipid able to bind phosphate, by two different procedures. One of them, generally used for lipid extraction, leads to the preparation of a more active crude proteolipid. This crude proteolipid has been purified by various chromatographic procedures and the active fraction, in phosphate binding, is always associated with cardiolipin. Its molecular weight seems to be close to 10000. The phosphate binding shows ligand saturation behavior and is inhibited by arsenate and N-ethylmaleimide; succinate is noninhibitory. This protein seems to be dependent on the mitoribosomal synthesis since it is not present in mitochrondria of mutant "petite colonie" and its amount largely decreases in mitochondria from yeast grown in the presence of chloramphenicol. It is possible to extract a proteolipid from the oligomycin sensitive ATPase, showing the same activity and properties. The hypothesis that this proteolipid acts as a part of the Pi carrier and constitutes the oligomycin-sensitive ATPase complex is discussed.

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