Photoaffinity labelling of the purine-cytosine permease of Saccharomyces cerevisiae

8-Azidoadenine was used as a photoaffinity reagent to characterize the purine-cytosine permease of Saccharomyces cerevisiae. It is a potent competitive inhibitor of cytosine uptake and irradiation of the cells incubated with the label induced the irreversible inactivation of cytosine uptake. Addition of excess cytosine prevented this labelling which was restricted to the outer face of the plasma membrane since it was not accumulated by the cells. In the strain with the amplified purine-cytosine permease gene the maximum cytosine uptake rate was increased 4-5-fold relative to wild type without a modification of the Michaelis constant of uptake (Kt); no uptake could be measured in the deleted strain. The relative amounts of specific labelling determined for the cells and for membrane preparations were 0, 1 and 4 for the null, the wild-type and the amplified strains, respectively. One major band specifically labelled by [3H]azidoadenine, corresponding to a polypeptide with an apparent molecular mass of 45 kDa, was observed in the wild type, amplified in the strain carrying the multicopy plasmid and not detected in the deleted strain. Therefore this polypeptide corresponds to the purine-cytosine permease.     

Screening of an intragenic second-site suppressor of purine-cytosine permease from Saccharomyces cerevisiae. Possible role of Ser272 in the base translocation process.

The purine-cytosine permease from Saccharomyces cerevisiae mediates the active transport through the plasma membrane of adenine, hypoxanthine, guanine and cytosine using the proton electrochemical potential difference as an energy source. Analysis of the activity of strains mutated in a hydrophilic segment (371-377) of the polypeptidic chain has shown the involvement of this segment in the maintenance of the active three-dimensional structure of the carrier. In an attempt to identify permease domains that could interact functionally and/or physically with this segment, we looked for second-site mutations that could suppress the effects of amino acid changes in this region. This paper describes a positive screen that has allowed the isolation of one suppressor from a permease mutant displaying the N374I change (fcy2-20 allele), a substitution that induces a dramatic decrease in the affinity of the carrier for adenine, cytosine and hypoxanthine. The second-site mutation corresponds to the replacement of the Ser272 residue by Leu. Its suppressive effect is shown to be a partial restoration of the binding of cytosine and hypoxanthine to the permease. To test whether this second-site mutation is specific for the fcy2-20 allele, two double mutants were constructed (Fcy2pT213I, S272L and Fcy2pS272L, N377G). Results obtained with these two double mutants showed that the suppressive effect of S272 L replacement was not specific for the original N374I change. To understand the general effect of this amino acid replacement for the three distinct double mutants, a strain overexpressing Fcy2pS272I, was constructed. Kinetic analysis of this strain showed that, by itself, the S272 L change induced an improvement in the base-binding step that could account for its global suppressive effect. Moreover, S272 L induced a decrease in the turnover of the permease, thus showing the involvement of S272 in the translocation process. Taking into account the topological model of the permease proposed here, this Ser residue is probably located in a transmembrane amphipathic alpha-helix (TM5). The location and the observed decrease in the turnover of the carrier observed with the S272 L change lead us to propose that S272 could be part of a hydrophilic pore involved in the translocation of the base and/or the proton.     

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.     

In vivo and in vitro studies of the purine-cytosine permease of Saccharomyces cerevisiae. Functional analysis of a mutant with an altered apparent transport constant of uptake.

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. cerevisiae 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 (Ktapp) and Vmax] and on plasma-membrane preparations, by measurements of binding parameters at equilibrium [(Kd and maximum amount of binding sites/Bmax)]. For strain pAB4, the Ktapp and Vmax of uptake were almost similar for all solutes considered [1.8-2.6 microM and 8.5-10.2 nmol.min-1.(10(7) cells)-1]. The main effect of the mutations in strain pAB25 was based on a large increase in Ktapp for all ligands except adenine. Plasma membranes of each strain displayed one class of specific binding sites. Variations in Kd of 0.4-1 microM were observed for pAB4. These slight variations had no effect on the Ktapp of uptake measured for the corresponding solutes. In contrast, using pAB25 membranes, Kd increased dramatically; 2.6 microM, 40 microM and 96 microM for adenine, cytosine and hypoxanthine, respectively. These increments were correlated to variations in Ktapp of the uptake for cytosine and hypoxanthine. Therefore, we conclude that modification in the Ktapp 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.     

GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae.

The gene encoding the galactose permease of Saccharomyces cerevisiae (GAL2) was cloned. The clone restores galactose permease activity to gal2 yeasts and is regulated by galactose in a manner similar to other GAL gene products (GAL1, -7, and -10). Experiments with temperature-conditional secretory mutants indicated that transport of the GAL2 gene product to the cell surface requires a functional secretory pathway. In addition, gene fusions were constructed between the GAL2 gene and the Escherichia coli lacZ gene. The GAL2-lacZ gene fusions code for galactose-regulated beta-galactosidase activity in yeasts. The beta-galactosidase activity was found to be membrane bound.     

Evidence for a new chromosome in Saccharomyces cerevisiae.

The current yeast map has 16 chromosomes, each originally defined by a centromere-linked gene unlinked to previously defined centromere markers. We examined four genes, cly2, KRB1, AMY2, and tsm0115, each centromere linked, but previously thought to be not on chromosomes I to XVI. We found that AMY2 is linked to cly2, and both are on chromosome II. tsm0115 is on the left arm of chromosome XVI. We confirm the earlier evidence that KRB1 is not on chromosomes I through XVI. This gene thus defines a new chromosome XVII. We also report meiotic linkage of met4 and pet8 (on chromosome XIV), confirming the connection between the petx-kex2 fragment of XIV and the centromere of XIV.     

Induction and inhibition of the allantoin permease in Saccharomyces cerevisiae.

Allantoin uptake in Saccharomyces cerevisiae is mediated by an energy-dependent, low-Km, active transport system. However, there is at present little information concerning its regulation. In view of this, we investigated the control of alloantoin transport and found that it was regulated quite differently from the other pathway components. Preincubation of appropriate mutant cultures with purified allantoate (commercial preparations contain 17% allantoin), urea, or oxalurate did not significantly increase allantoin uptake. Preincubation with allantoin, however, resulted in a 10- to 15-fold increase in the rate of allantoin accumulation. Two allantoin analogs were also found to elicit dramatic increases in allantoin uptake. Hydantoin and hydantoin acetic acid were able to induce allantoin transport to 63 and 95% of the levels observed with allantoin. Neither of these compounds was able to serve as a sole nitrogen source for S. cerevisiae, and they may be non-metabolizable inducers of the allantoin permease. The rna1 gene product appeared to be required for allantoin permease induction, suggesting that control was exerted at the level of gene expression. In addition, we have shown that allantoin uptake is not unidirectional; efflux merely occurs at a very low rate. Allantoin uptake is also transinhibited by addition of certain amino acids to the culture medium, and several models concerning the operation of such inhibition were discussed.     

Gene-enzyme relationships in the proline biosynthetic pathway of Saccharomyces cerevisiae.

The PRO1, PRO2, and PRO3 genes were isolated by functional complementation of pro1, pro2, and pro3 (proline-requiring) strains of Saccharomyces cerevisiae. Independent clones with overlapping inserts were isolated from S. cerevisiae genomic libraries in YEp24 (2 microns) and YCp50 (CEN) plasmids. The identity of each gene was determined by gene disruption, and Southern hybridization and genetic analyses confirmed that the bona fide genes had been cloned. Plasmids containing each gene were introduced into known bacterial proline auxotrophs, and the ability to restore proline prototrophy was assessed. Interspecies complementation demonstrated that the S. cerevisiae PRO1 gene encoded gamma-glutamyl kinase, PRO2 encoded gamma-glutamyl phosphate reductase, and PRO3 encoded delta 1-pyrroline-5-carboxylate reductase. The presence of the PRO3 gene on a high-copy-number plasmid in S. cerevisiae caused a 20-fold overproduction of delta 1-pyrroline-5-carboxylate reductase. The PRO2 gene mapped on chromosome XV tightly linked to cdc66, and the PRO3 gene was located on the right arm of chromosome V between HIS1 and the centromere.     

The decisive role of the Saccharomyces cerevisiae cell cycle behaviour for dynamic growth characterization.

The dynamic behaviour of the cell cycle and the physiology of Saccharomyces cerevisiae was monitored in transient experiments. Frequent flow cytometric analyses of the DNA (nuclear phase state) and the cell size enabled us to characterize the proliferation properties of yeast cells under well controlled and undisturbed cultivation conditions. Preliminarily, the correlation between flow cytometric light scattering measurements and the cell size was attested for yeasts. These flow cytometric results are compared with the physiological behaviour of the culture that was detected by high resolution on-line analyses and off-line measurements. The presented results focus on the importance of the yeast cell cycle behaviour for the dynamic growth characterization. Any kind of transients in yeast cultures induced partial synchronization. The characteristics and the time course of the yeast cell cycle were found to be strongly dependent on the physiological environment.     

Genetics and physiology of proline utilization in Saccharomyces cerevisiae: enzyme induction by proline.

Proline is converted to glutamate in the yeast Saccharomyces cerevisiae by the sequential action of two enzymes, proline oxidase and delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase. The levels of these enzymes appear to be controlled by the amount of proline in the cell. The capacity to transport proline is greatest when the cell is grown on poor nitrogen sources, such as proline or urea. Mutants have been isolated which can no longer utilize proline as the sole source of nitrogen. Mutants in put1 are deficient in proline oxidase, and those in put2 lack P5C dehydrogenase. The put1 and put2 mutations are recessive, segregate 2:2 in tetrads, and appear to be unlinked to one another. Proline induces both proline oxidase and P5C dehydrogenase. The arginine-degradative pathway intersects the proline-degradative pathway at P5C. The P5C formed from the breakdown of arginine or ornithine can induce both proline-degradative enzymes by virtue of its conversion to proline.     

Isolation and preliminary characterization of Saccharomyces cerevisiae proline auxotrophs.

Proline-requiring mutants of Saccharomyces cerevisiae were isolated. Each mutation is recessive and is inherited as expected for a single nuclear gene. Three complementation groups cold be defined which are believed to correspond to mutations in the three genes (pro1, pro2, and pro3) coding for the three enzymes of the pathway. Mutants defective in the pro1 and pro2 genes can be satisfied by arginine or ornithine as well as proline. This suggests that the blocks are in steps leading to glutamate semialdehyde, either in glutamyl kinase or glutamyl phosphate reductase. A pro3 mutant has been shown by enzyme assay to be deficient in delta 1-pyrroline-5-carboxylate reductase which converts pyrroline-5-carboxylate to proline. A unique feature of yeast proline auxotrophs is their failure to grown on the rich medium, yeast extract-peptone-glucose. This failure is not understood at present, although it accounts for the absence of proline auxotrophs in previous screening for amino acid auxotrophy.     

The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

In response to nitrogen starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. This dimorphic transition is regulated by the Galpha protein GPA2, by RAS2, and by elements of the pheromone-responsive MAP kinase cascade, yet the mechanisms by which nitrogen starvation is sensed remain unclear. We have found that MEP2, a high affinity ammonium permease, is required for pseudohyphal differentiation in response to ammonium limitation. In contrast, MEP1 and MEP3, which are lower affinity ammonium permeases, are not required for filamentous growth. Deltamep2 mutant strains had no defects in growth rates or ammonium uptake, even at limiting ammonium concentrations. The pseudohyphal defect of Deltamep2/Deltamep2 strains was suppressed by dominant active GPA2 or RAS2 mutations and by addition of exogenous cAMP, but was not suppressed by activated alleles of the MAP kinase pathway. Analysis of MEP1/MEP2 hybrid proteins identified a small intracellular loop of MEP2 involved in the pseudohyphal regulatory function. In addition, mutations in GLN3, URE2 and NPR1, which abrogate MEP2 expression or stability, also conferred pseudohyphal growth defects. We propose that MEP2 is an ammonium sensor, generating a signal to regulate filamentous growth in response to ammonium starvation.     

Inactivation of maltose permease and maltase in sporulating Saccharomyces cerevisiae

Maltose transport and maltase activities were inactivated during sporulation of a MAL constitutive yeast strain harboring different MAL loci. Both activities were reduced to almost zero after 5 h of incubation in sporulation medium. The inactivation of maltase and maltose permease seems to be related to optimal sporulation conditions such as a suitable supply of oxygen and cell concentration in the sporulating cultures, and occurs in the fully derepressed conditions of incubation in the sporulation acetate medium. The inactivation of maltase and maltose permease under sporulation conditions in MAL constitutive strains suggests an alternative mechanism for the regulation of the MAL gene expression during the sporulation process.