Determination of a specific region of the purine–cytosine permease involved in the recognition of its substrates

Three u.v.-induced mutants of the purine–cytosine permease gene of Saccharomyces cerevisiae, with altered apparent Michaelis constant of transport (K_mapp), were cloned and sequenced. One of the mutants had extensive nucleotide replacement, whereas the other two had a single mutation. To evaluate the contribution of the different amino acid replacements to the phenotype of the complex mutant, simpler mutants were created by site-directed mutagenesis. All the amino acid replacements found in the segment from amino acids 371 to 377 inclusive, contribute to the determination of the phenotype. According to the model postulated this segment lies on the cell surface. In particular, amino acids at position 374 and 377 modulate the affinity of the permease towards its substrates. In the wild-type, when asparagine is present at both of these positions, the lowest K_mapp values are found.