M. R. Chevallier

The purine-cytosine permease gene of Saccharomyces cerevisiae: primary structure and deduced protein sequence of the FCY2 gene product

A 2.1 kb DNA segment carrying the purine‐cytosine permease gene (FCY2) of Saccharomyces cerevisiae was sequenced, the primary structure of the protein (533 amino acids) deduced and a folding pattern in the membrane is proposed for the permease protein. Expression of the FCY2 gene product requires a functional secretory pathway and is reduced in mnn9, a mutant strain deficient in outer chain glycosylation. The FCY2 gene was mapped on the right arm of chromosome V close to the HIS1 gene.

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 (Kmapp), 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 Kmapp values are found.

Evolutionary relationship and secondary structure predictions in four transport proteins ofSaccharomyces cerevisiae

SummaryThe comparison of the amino acid sequences of four yeast transport proteins indicates that there is a questionable relatedness between the uracil permease (FUR4) and the purine-cytosine permease (FCY2), whereas the arginine permease (CAN1) and the histidine permease (HIP1) clearly originated from a common molecular ancestor. The analysis of the primary structure of these transport proteins by two methods of secondary structure predictions suggests the presence of 9–12 membrane-spanning α-helices in each polypeptide chain. These results are concordant in that 90% of the α-helices were determined by both methods to be at the same positions. In the aligned sequencesHIP1 andCAN1, the postulated membrane-spanning α-helices often start at corresponding sites, even though the overall sequence similarity of the two proteins is only 30%. In the aligned DNA coding sequences ofCAN1 andHIP1, synonymous nucleotide substitutions occur with very similar frequencies in regions where the replacement substitution (changing the amino acids) frequencies are widely different. Moreover, our data suggest that the replacement substitutions can be considered as neutral in the N-terminal segment, whereas the other regions are subject to a conservative selective pressure because, if compared to a random drift, the replacement substitutions are underrepresented.

The Uracil Permease of Schizosaccharomyces pombe: A Representative of a Family of 10 Transmembrane Helix Transporter Proteins of Yeasts

The uracil permease gene of Schizosaccharomyces pombe was cloned and sequenced. The deduced protein sequence shares strong similarities with five open reading frames from Saccharomyces cerevisiae, namely the uracil permease encoded by the FUR4 gene, the allantoin permease encoded by DAL4, a putative uridine permease (YBL042C) and two unknown ORFs YOR071c and YLR237w.

Transport properties of a C. albicans amino-acid permease whose putative gene was cloned and expressed in S. cerevisiae

Using a gene bank of C. albicans, the lysine-permease deficiency in a strain of S. cerevisiae was complemented, and the restriction map of the corresponding C. albicans DNA fragment was constructed. Its expression in S. cerevisiae showed that the permease of C. albicans actively transports arginine (KT=18 μmol/l, Jmax=26 nmol/min per mg dry weight), lysine (KT=12 μmol/l, Jmax=18 nmol/min per mg dry weight), histidine (KT=37 μmol/l, Jmax=9.7 nmol/min per mg dry weight), as well as their toxic analogues canavanine and thialysine, with high affinity. The intracellular concentration of basic amino acids transported into S. cerevisiae by the C. albicans permease reaches more than a thousand-times-higher value compared to the external concentration in the medium. Accumulated amino acids do not leave the cells. The uptake is strongly reduced by the protonophores and inhibitors of plasma membrane H+-ATPase.

Candida albicans geneCAN1 is highly homologous to other yeast andE. coli genes coding for amino acid permeases

The amino acid permease C a n l of Candida albicans transports arginine, lysine and histidine [1] with a high affinity. The CAN1 gene (1713 bp) coding for this permease was cloned as a D N A fragment (3385 bp) complement ing the lysine-permease deficiency in S. cerevisiae. Protein C a n l is 571 amino acids long, with a calculated molar mass of 63 343 Da. Analysis of its deduced primary structure revealed ten membrane-spanning segments and three potential N-glycosylation sites [2]. The protein sequence is strongly similar to both permeases for basic amino acids of Saccharomyces cerevisiae C a n l and Lyp l [3, 4], and to the lysine-specific permease LysP of E. coli [5]. The detailed comparison of amino acid sequences of all three yeast permeases for basic amino acids showed that C. albicans Can1 shares 57.3 % identical (75.5 % similar) residues with S. cerevisiae Can1 and 49.7 % identity (73.3 % similarity) with Lyp l permease. The number and distribution of amino acid residues conserved in all three permeases are summarized in Table I.

Chromosomal mapping of the uracil permease gene of Saccharomyces cerevisiae

SummaryThe gene FUR4, coding for the uracil permease in Saccharomyces cerevisiae, was mapped on chromosome II, at a distance of 7.8 cM from the centromere on the right arm of the chromosome. In a first step, we used the chromosome loss mapping method developed by Falco and Botstein (1983) to determine on which chromosome the gene mapped. After the observation that FUR4 was closely linked to GAL10, one of the three genes forming the gal cluster (Bassel and Mortimer 1971), we could determine precisely the position of the gene on chromosome II.