Evidence for a dynamic role for proline376 in the purine-cytosine permease of Saccharomyces cerevisiae.
The purine-cytosine permease (PCP), a carrier located in the plasma membrane of Saccharomyces cerevisiae, mediates the active transport of purine (adenine, guanine and hypoxanthine) and cytosine into the cell. Previous studies [Ferreira, T, Brèthes, D., Pinson, B., Napias, C. & Chevallier, J. et al. (1997) J. Biol. Chem. 272, 9697-9702] suggest that the hydrophilic segment 371-377 (-I-A-N-N-I-P-N-) of the polypeptide chain may play a key role in the correct three-dimensional structure of the active carrier. This paper describes the effects of mutations in this particular segment: a four-residue deletion, Delta374-377, and two substitutions, P376G and P376R. The Delta374-377 PCP was expressed in tiny amounts and was totally inactive. When compared with the wild-type, the P376G PCP showed slightly decreased amounts and was able to transport the bases with significantly increased affinity and decreased turnover. The P376R PCP was normally expressed and targeted to the plasma membrane; however, despite a normal number of base-binding sites [1000-1200 pmol.(mg protein)-1], this mutated carrier was completely unable to transport any of its ligands. In addition, the Kd(app) for hypoxanthine binding was completely independent of the pH (within the range 3.5-6.0), showing that the conformational change induced by ligand binding was no longer present. Our results show that the 374-377 segment is essential for the expression and activity of this carrier. They also show that the P376 residue is part of an unusual secondary structure, probably a beta-turn motif, which must play a crucial dynamic role in the translocation process.
Ferreira
T
T
Institut de Biochimie et Géntique Celluaires de Centre National de la Recherche Scientifique, UPR 9026, Bordeaux, France.
Napias
C
C
Chevallier
J
J
Brèthes
D
D
D000225:Adenine / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003596:Cytosine / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D019271:Hypoxanthine / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D033702:Nucleobase Transport Proteins; D017354:Point Mutation; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D002352:Carrier Proteins; D004271:DNA, Fungal; C043181:FCY2 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033702:Nucleobase Transport Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D019271:Hypoxanthine; D003596:Cytosine; D011392:Proline; D000225:Adenine