Amino Acid Transport in a Polyaromatic Amino Acid Auxotroph of Saccharomyces cerevisiae

Transcription

1 JOURNAL OF BACTERIOLOGY, Sept. 1973, p Copyright O 1973 American Society for Microbiology Vol. 115, No. 3 Printed in U.S.A. Amino Acid Transport in a Polyaromatic Amino Acid Auxotroph of Saccharomyces cerevisiae RANDOLPH L. GREASHAM' AND ALBERT G. MOAT Department of Microbiology, Hahnemann Medical College, Philadelphia, Pennsylvania Received for publication 28 May 1973 The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing "4C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for L-amino acids and a Km ranging from 1.5 x 10-5 to 5.0 x 10-5 M. Optimal transport activity occurred at ph 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect. A mutant of Saccharomyces cerevisiae auxotrophic for tryptophan, phenylalanine, and tyrosine was isolated by selection techniques described previously (11, 12). The initiation of growth of this auxotroph was inhibited by several amino acids but most effectively by isoleucine, threonine, valine, and aspartic acid (A. G. Moat and A. Kateiva, Bacteriol. Proc., p. 119, 1968). The prototrophic parent was unaffected by the addition of these amino acids to the growth medium. It was considered that these amino acids might inhibit the initiation of growth of the auxotrophic strain through the competitive inhibition of the uptake of the required amino acids. Halvorson and Cohen (7), Surdin, et al. (16), and Grenson, et al. (4) demonstrated an amino acid transport system in yeast which is responsible for the transport of all neutral as well as basic amino acids. However, some yeasts have been shown to have specific transport systems for arginine and lysine (2, 5). In S. chevalieri, Schwencke and Schwencke (14) observed the presence of a specific proline transporting system. A systematic study of the transport of amino acids by the aromatic amino acid auxotroph of S. cerevisiae and its prototrophic parent revealed that both strains possess a general amino acid transport system for neutral and basic amino acids. MATERIALS AND METHODS Organisms. A haploid strain of Saccharomyces cerevisiae 174/1D (11, 12) and a mutant auxotrophic for tryptophan, tyrosine, and phenylalanine derived from it (A. G. Moat and A. Kateiva, Bacteriol. Proc., p. 119, 1968) were used in these investigations. Both strains were grown aerobically at 30 C in a minimal defined medium (15) buffered at ph 5.6. This medium was supplemented with tryptophan, tyrosine, and phenylalanine at final concentrations of 10-4 M for the aromatic mutant. Chemicals. L-tryptophan-3- "C (specific activity, 23 mci/mmol), DL-leucine-l-1-C (specific activity, 2.56 mci/mmol) and uniformly labeled "C-L-isoleucine (specific activity, 236 mci/mmol) were purchased from New England Nuclear Corp., Boston, Mass. Cycloheximide was purchased from Nutritional Biochemical Corp., Cleveland, Ohio. Amino acid transport. A sample of the cell suspension was added to 25 ml of 0.1 M sodium phosphate buffer (ph 5.7) plus 2% glucose to bring the final cell concentration to 0.45 mg (dry wt)/ml. After preincubation for 1.5 h at 32 C in a rotary water bath shaker, "4C-labeled amino acid and any nonlabeled test amino acid(s) were added. Two 2-ml samples were removed from the reaction mixture at 1-min intervals. One sample was immediately transferred I Present address: Research Department, Commercial Solvents Corp., Terre Haute, Ind ferred into 10 ml of 10% trichloroacetic acid. The into 10 ml of water at 4 C and the other was trans- 975

2 976 GREASHAM AND MOAT J. BACTERIOL. former sample was deposited on a membrane filter (Gelman type GA-6, 0.45 Am, 47 mm diameter), washed with 24 ml of ice water, and glued to a planchet, and the radioactivity was determined by using a Baird-Atomic thin-window gas-flow counter. Radioactivity was assessed to a level of statistical accuracy (usually at least 2,000 counts). The trichloroacetic acid-treated sample was similarly filtered and counted to determine the amount of amino acid incorporated into protein. RESULTS Effect of amino acids on growth. The growth of S. cerevisiae 174/1D and the polyaromatic amino acid auxotroph was tested in the presence of various amino acids. Of the 19 amino acids tested, isoleucine, threonine, aspartic acid, serine, and valine exhibited substantial inhibition of the initiation of growth of the mutant (Fig. 1). Combinations of these amino acids have been shown to exert still greater effects on the ability of the mutant to initiate growth, but no comparable inhibition of growth was observed with the parental strain (A. G. Moat and A. Kateiva, Bacteriol. Proc., p. 119, 1968). Tryptophan uptake in exponential phase >-. ISOLEUCINE I.- Tjjo.G z 0. 0: C) 0 4 a zo.c.aspartate z W&I 0 a. 0 cells. Alteration in the rate of amino acid transport during the exponential phase of growth was examined by using "4C-labeled tryptophan. All experiments were conducted at ph 5.7 because preliminary investigation revealed that optimal tryptophan uptake occurred at this ph. Samples were removed from growing cultures of the aromatic auxotroph at various times during the exponential phase and the rate of tryptophan uptake was determined. The rate of uptake increased to a maximum at an optical density of approximately 0.3 (at 600 nm) at mid-exponential phase (Fig. 2). For this reason, all subsequent investigations on amino acid uptake were performed with mid-exponential phase cells. Tryptophan uptake began immediately after the addition of tryptophan to the reaction mixture and continued at a linear rate for 4 min and then leveled off (Fig. 3). To assess the ability of cells to maintain the intracellular concentration of tryptophan, cells were added to the reaction mixture and allowed to incubate for 5 min and were then washed three times with 0.1 M sodium phosphate buffer (ph 5.7). After the third washing, no detectable amount of 14C-tryptophan was found in the washings. 0.I 0.4 ~ ol0.. o.s THREONINE 0 4 VALINE i-f I a FIG. 1. Inhibitory effect of isoleucine, threonine, valine, and aspartic acid on the initiation of growth of a polyaromatic auxotroph of S. cerevisiae strain 1 74/1D. An inoculum culture of the mutant was grown for 18 h on complete medium, washed twice with sterile distilled water, and diluted to yield a suspension with an optical density of 0.05 at 600 nm. An inoculum of 0.1 ml was used to inoculate 5 ml of defined medium (15) supplemented with: 0, tryptbphan (25 Ag/ml), tyrosine, and phenylalanine (15 jug/ml each); 1, aromatic amino acids plus isoleucine (88 ug/ml), threonine (79 Ag/ml), valine (70,g/ml), and aspartic acid (200 ug/ml); 2, aromatic amino acids plus twice the concentration of each additional amino acid; and 3, aromatic amino acids plus three times the concentration of each amino acid.

3 VOL. 115, 1973 S. CEREVISIAE AMINO ACID TRANSPORT TIMIE- HOURS FIG. 2. Rate of L-tryptophan uptake in cell samples which were removed at different time periods during the growth of an aromatic auxotrophic mutant of S. cerevisiae strain 1 74/1D. The uptake system contained 0.9 mg (dry weight) of preincubated cells suspended in 2 ml of 0.1 M sodium phosphate buffer (ph 5.7) containing 2o glucose, 10-4M L-tryptophan, and 1.47 x 10-' M L-tryptophan-3-'4C. Tryptophan uptake was stopped after 3 min by adding 10 ml of water at 4 C and filtering. Symbols: 0, growth; 0, tryptophan uptake. The cells were then resuspended in buffer containing 2% glucose and allowed to incubate for 15 min. Samples were removed periodically and analyzed for the amount of intracellular 14Ctryptophan. Maintenance of a constant intracellular concentration of tryptophan suggested that the transport of tryptophan was unidirectional. The initial fate of the transported tryptophan is also shown in Fig. 3. Essentially no "4C-tryptophan was found in the trichloroacetic acid-precipitable fraction, indicating that tryptophan initially enters the intracellular metabolic pool and is not immediately incorporated into protein. This might be expected since incorporation into protein would require the addition of a full complement of amino acids. Regulation of amino acid uptake. Schwencke and Schwencke (14) found that the uptake of proline by S. chevalieri was enhanced by incubating the cells in nitrogen-free medium containing galactose as the only source of energy. The rate of tryptophan uptake by S. cerevisiae increased with increasing length of preincubation in a nitrogen-free buffer containing 2% glucose (Table 1). This suggested that the endogenous metabolic pool may regulate the uptake of tryptophan. To investigate this possibility further, cells were preloaded with both tryptophan and leucine for 6 min. The cells were then washed three times with 0.1 M sodium phosphate buffer (ph 5.7) and resuspended in the reaction mixture, and tryptophan uptake. was determined. Cells preloaded with leucine and tryptophan showed an 83 to 93% inhibition of tryptophan uptake (Fig. 4). These results suggested that tryptophan uptake is influenced by saturation of the amino acid pool with other amino acids as well as tryptophan. Additional supporting evidence for this concept was obtained by examining the effect of the intracellular metabolic pool size on tryptophan uptake. Four 200-mg (dry weight) samples were taken. The first and second samples were taken before and after preincubation with 2% glucose. The third and fourth samples were taken after : 25 TOTAL UPTAKE C 20 C_ 15 -E 10 C= X_ C= /PROTEIN INCORPORATION U FIG. 3. Time course of L-tryptophan uptake and incorporation into protein by an aromatic auxotrophic mutant of S. cerevisiae strain 174/iD. The system contained 0.45 mg (dry weight) of cells per ml of 0.1 sodium in phosphate buffer (ph 5.7) containing 2% glucose, 10- ML-tryptophan, and 1.75 x 10-7 M L-tryptophan-3-14C. Radioactivity associated with the trichloracetic acid-insoluble material is representative of protein incorporation. TABLE 1. Effect of preincubation of cells in the presence of glucose on the rate of 'L-tryptophan uptake by S. cerevisiae mutant Preincubation (min) L-Tryptophan uptakea aexpressed as nanomoles per minute per milligram of (dry weight) cells.

4 978 GREASHAM AND MOAT J. BACTERIOL X = FIG. 4. Effect of preloading the amino acid pool on L-tryptophan uptake by a polyaromatic auxotroph of S. cerevisiae. Cells were preincubated for 6 min in the presence of L-tryptophan and L-leucine, both at 10-4 M. Uptake conditions were as in Fig. 3. Symbols: 0, no preloading; 0, preloading with L-leucine; A, preloading with tryptophan. the cells had been preincubated and then incubated in the presence of 10-4 M tryptophan for 5 and 10 min. The amount of each of the various amino acids in the extractable pool was determined by previously described methods (10). The amino acids in the extractable pool remained fairly constant after the addition of tryptophan (Table 2). Tryptophan was not detectable in the pool during preincubation, but began to accumulate immediately after its addition. In Table 1, the rate of tryptophan uptake was shown to increase about 87% after 90 min of preincubation. The amount of tryptophan present in the intracellular pool before and after 90 min of preincubation was nil, indicating that the amounts of other amino acids present in the pool definitely affected the amount of tryptophan uptake. Effect of metabolic inhibitors on amino acid uptake. Wiley and Matchett (Bacteriol. Proc., p. 106, 1967) reported that protein synthesis was required for the maintenance of the tryptophan uptake system in Neurospora. Contrarily, Grenson et al. (3) presented data which suggested that concomitant protein synthesis was not required for the maintenance of the uptake system in yeast. Their findings suggested that the intracellular concentration of amino acids resulting from the inhibition of protein synthesis by cycloheximide was the responsible inhibitor of the transport system. To determine which of these interpretations could be applied to the uptake system in the aromatic auxotroph of S. cerevisiae, cycloheximide, known to inhibit protein synthesis in yeast (9), was shown to inhibit tryptophan uptake in growing cells after 30 min. However, cycloheximide had essentially no effect on the rate of L-tryptophan uptake by resting cells (Fig. 5). These results suggest that protein synthesis is not a direct requirement for maintenance of the amino acid transport system in yeast. As shown in Fig. 5, 2,4-dinitrophenol at a concentration of 5.6 x 10-i M was an effective inhibitor of tryptophan uptake, providing convincing evidence that the amino acid uptake system is energy dependent. Specificity of the amino acid transport system. The rate of L-tryptophan uptake was essentially the same in both the wild and mutant strains of S. cerevisiae (Fig. 6). It was of interest to determine the specificity of the amino acid transport system in both strains by measuring tryptophan uptake in the presence of various amino acids. The results presented in Table 3 show that tryptophan transport was inhibited by several amino acids with only slight differences being exhibited in the wild type as compared to the auxotroph. These findings indicate that a general amino acid TABLE 2. Effect ofpreincubation with glucose on the concentration of amino acids in the endogenous pool before and after 14C-tryptophan uptake by S. cerevisiae mutant Concna Preincu- Preincubation bation Amino acid Before After plus 5 plus 10 min preincuof inpreincumin of inbation bation cubation cubation with tryp- with tryptophan at tophan at 10-4M 10-4M Aspart-ic- acid Threonine Serine Glutamic acid Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Tryptophan Histidine Arginine a weight) Expressed cells. as nanomoles per milligram of (dry

5 VOL. 115, 1973 S. CEREVISIAE AMINO ACID TRANSPORT 979, 25- TRYPTOPHAN UPTAKE cc E 10 CONTROL CYCLOHEXIMIDE ADDED DNP ADDED FIG. 5. Effect of cycloheximide and 2,4-dinitrophenol (DNP) on L-tryptophan uptake by S. cerevisiae mutant. Cycloheximide and 2,4-dinitrophenol were added at concentrations of 7.1 x 10-6 M and 5.6 x 10-4 M, respectively. Uptake conditions were as in Fig C=,WILD Z15 - C=d C. 5 - MUTANT FIG. 6. Comparison of L-tryptophan uptake in both wild-type and mutant strains of S. cerevisiae. Conditions were as in Fig. 3. transport system is operative in both strains. Inhibition of L-tryptophan uptake by the basic amino acids L-arginine, L-histidine, and L-lysine suggested that the general system includes the basic amino acids. Since a few amino acids (L-alanine, L-proline, L-aspartic acid, and L- glutamic acid) showed little or no inhibition in either the mutant or the wild type, it was concluded that these amino acids enter the cell by a separate transport system. The general amino acid transport system was observed to be stereospecific as shown by the lack of an inhibitory effect by the D-isomers of leucine and isoleucine (Table 3). As stated previously, L-histidine exhibited a high degree of inhibition of L-tryptophan uptake. L-histamine at a concentration of 10-i M exerted no effect, suggesting that the a-carboxyl group is necessary for amino acid recognition at the transport site. Kinetic studies. The kinetics of the amino acid transport system were also investigated (Fig. 7). Saturation kinetics of the uptake of L-tryptophan was observed with increasing concentrations of exogenous tryptophan. The apparent Km (affinity constant) for L-tryptophan uptake ranged from 1.5 x 10-I to 5 x 10-5 M. At a concentration of 10-4 M, tryptophan uptake was competitively inhibited by other amino acids as indicated by the common intercept of the inhibitor and control plots. The apparent inhibition constants (K,) of each of the inhibitory amino acids were determined. The results (Table 4) suggest that the uptake of amino acids is enzymatic in nature and that L-tryptophan as TABLE 3. Inhibition of the initial velocity of uptake of L-tryptophan by amino acids at 10-4 M concentration in both the wild-type and mutant strains of S. cerevisiae Amino acid Mutant tion (%) inhibi- Wild bition type (%) inhi- L-Arginine L-Histidine L-Leucine L-Lysine L-Phenylalanine L-Tyrosine L-Cysteine L-Glutamine L-Valine L-Isoleucine L-Serine DL-Methionine L-Threonine L-Asparagine L-Glycine 6 12 L-Alanine 0 9 L-Proline 0 6 L-Aspartic acid 0 3 L-Glutamic acid 0 6 D-Leucine 0 D-Isoleucine 0

6 980 GREASHAM AND MOAT J. BACTERIOL. V o. [RiP (0-5) FIG. 7. Competitive inhibition of L-tryptophan uptake by various amino acids in the mutant strain of S. cerevisiae. All amino acids were added at a concentration of 10-4 M. The uptake conditions were as described in Fig. 2. V is expressed in nanomoles per minute per milligram (dry weight) of cells. Control represents uptake in the absence of an inhibitor. TABLE 4. Competitive inhibition of L-tryptophan uptake by several amino acids in S. cerevisiae mutant Amino acid Inhibition constant (M) L-Histidine 4.7 x 10-' L-Arginine 5.3 x 10-6 L-Leucine 9.5 x 10-6 L-Lysine 1.0 x 10-s L-Phenylalanine 2.0 x 10-' L-Cysteine 2.5 x 10-s L-Tyrosine 3.0 x 10-5 L-Valine 3.8 x 10-s L-Glutamine 4.8 x 10-5 L-Isoleucine 5.6 x 10-5 DL-Methionine 5.6 x 10-' L-Serine 6.9 x 10-' L-Threonine 9.2 x 10-' well as the inhibitory amino acids are transported via a common transport system. Effect of tryptophan on the uptake of isoleucine and leucine. If the amino acid transport system in both the mutant and wild-type strains represents a general amino acid transport system, then tryptophan should inhibit the uptake of other amino acids in both strains. With leucine and isoleucine as examples, transport of these amino acids was found to be inhibited by L-tryptophan (Table 5), providing further evidence that the amino acid transport system in both the aromatic amino acid auxotroph and the wild type is a general amino acid transport system for neutral as well as basic amino acids. 0 DISCUSSION The results presented clearly indicate that the polyaromatic amino acid auxotroph of S. cerevisiae and its wild-type parent possess a general amino acid transport system for neutral as well as basic amino acids. General amino acid transport systems with similar specificities have been demonstrated in Arthrobotrys conoides (6) and Neurospora crassa (17) as well as in various yeasts (4, 16, 17). In the filamentous fungus Penicillium chrysogenum, acidic amino acids are also transported via a general amino acid transport system (8). The amino acid transport system in S. cerevisiae exhibited a rate of L-tryptophan uptake of 1.2 nmol per min per mg (dry wt) of cells in exponentially grown cells and approximately sevenfold higher activity after preincubation of cells for 2 h in a nitrogen-free buffer system containing 2% glucose. This increase in transport activity may be associated with the elimination of ammonium ions from the cell environment since Grenson et al. (4) reported that the general amino acid permease of yeast was inhibited by ammonium ions. However, the pool sizes of those amino acids which are transported by the same transport system as are the aromatic amino acids appear to decrease during the preincubation period (Table 2), indicating that the concentrations of these particular amino acids regulate L-tryptophan uptake. Supporting evidence was provided by the preloading experiments in which L-tryptophan as well as L-leucine restricted L-tryptophan uptake. Similarly, Crabeel and Grenson (1) found that histidine uptake was inhibited by the internal concentration of histidine in S. cerevisiae. This type of inhibition has been termed "transinhibition" by Ring et al. (13), although the exact mechanism of this inhibition is unknown. The amino acid transport system in S. cerevisiae was found to be independent of Effect of tryptophan on the uptake of TABLE 5. isoleucine and leucine by a polyaromatic amino acid auxotroph of S. cerevisiae Amino Aminoacd(1O'M) acid (1l-' aspecific Inhibition activitva (% L-Isoleucine-14C (U) 6.9 L-Isoleucine-'4C (U) L-tryptophan DL-Leucine-1-14C 3.7 DL-Leucine-1-14C L-tryptophan a Expressed as nanomoles per minute per milligram of (dry weight) cells.

7 VOL. 115, 1973 S. CEREVISIAE AMINO ACID TRANSPORT 981 protein synthesis. An indication of the stability of the transport system was derived from the finding that protein synthesis was minimal during preincubation studies. Also, since the activity of the transport system was unaffected by cycloheximide, it may be concluded that this system is relatively stable and differs markedly from the transport system in N. crassa which exhibits a very rapid turnover (W. R. Wiley and W. H. Matchett, Bacteriol. Proc., p. 106, 1967). Inhibition of the initiation of growth of the polyaromatic auxotroph of S. cerevisiae by certain amino acids may be explained, in part, on the basis of inhibition of uptake of the required amino acids. However, some differences between the degree of inhibition of uptake of the aromatic amino acids and the effect on growth remain to be explained. ACKNOWLEDGMENTS We thank Janet Finkbeiner and Alice Kurlans for their excellent technical assistance. This investigation was supported by National Science Foundation grant GB LITERATURE CITED 1. Crabeel, M., and M. Grenson Regulation of histidine uptake by specific feedback inhibition of two histidine permeases in Saccharomyces cerevisiae. Eur. J. Biochem. 14: Grenson, M Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. II. Evidence for a specific lysine-transporting system. Biochim. Biophys. Acta 127: Grenson, M., M. Crabeel, J. M. Wiame, and J. Bechet Inhibition of protein synthesis and simulation of permease turnover in yeast. Biochem. Biophys. Res. Commun. 30: Grenson, M., C. Hou, and M. Crabeel Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease. J. Bacteriol. 103: Grenson, M., M. Mousset, J. M. Wiame, and J. Bechet Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. I. Evidence for a specific arginine-transporting system. Biochim. Biophys. Acta 127: Gupta, R. K., and D. Pramer Amino acid transport by the filamentous fungus Arthrobotrvs conoides. J. Bacteriol. 103: Halvorson, H. O., and G. N. Cohen Incorporation des amino acids endogenes et exogenes dans les proteins de la levure. Ann. Inst. Pasteur 94: Hunter, D. R., and I. H. Segel Acidic and basic amino acid transport systems of Penicillium chrvsogenum. Arch. Biochem. Biophys. 144: Kerridge, D The effect of actidione and other antifungal agents on nucleic acid and protein synthesis in Saccharomvces carlsbergensis. J. Gen. Microbiol. 19: Moat, A. G., F. Ahmad, J. K. Alexander, and I. J. Barnes Alteration in the amino acid content of yeast during growth under various nutritional conditions. J. Bacteriol. 98: Moat, A. G., I. J. Barnes, and E. H. McCurley Factors affecting the survival of auxotrophs and prototrophs of Saccharomyces cerevisiae in mixed populations. J. Bacteriol. 92: Moat, A. G., N. Peters, Jr., and A. M. Srb Selection and isolation of auxotrophic yeast mutants with the aid of antibiotics. J. Bacteriol. 77: Ring, K., W. Gross, and E. Heinz Negative feedback regulation of amino acid transport in Streptomyces hydrogenans. Arch. Biochem. Biophys. 137: Schwencke, J., and N. Magana-Schwencke Derepression of a proline transport system in Saccharomyces chevalieri by nitrogen starvation. Biochim. Biophys. Acta 173: Snell, E. E., R. E. Eakin, and R. J. Williams A quantitative test for biotin and observations regarding its occurrence and properties. J. Amer. Chem. Soc. 62: Surdin, Y., W. Sly, J. Sire, A. M. Bordes, and H. derobichon-szulmajster Proprietes et contr6le genetique de systeme d'accumulation des acides amines chez Saccharomyces cerevisiae. Biochim. Biophys. Acta 107: Wiley, W. R., and W. H. Matchett Tryptophan transport in Neurospora crassa. I. Specificity and kinetics. J. Bacteriol. 92: