JOURNAL OF BACTERIOLOGY, Oct. 1975, p. 325-331 Copyright 0 1975 American Society for Microbiology
Vol. 124, Ng. 1 Printed in U.S.A.
Inhibition of Amino Acid Transport by Ammonium Ion in Saccharomyces cerevisiae ROBERT J. ROON,* FRED LARIMORE, AND JANE S. LEYY
Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 55455 Received for publication 25 June 1975
The rate of transport of L-amino acids by Saccharomyces cerevisiae e1278b increased with time in response to nitrogen starvation. This increase could be prevented by the addition of ammonium sulfate or cycloheximide. A slow time-dependent loss of transport activity was observed when ammonium sulfate (or ammonium sulfate plus cycloheximide) was added to cells after 3 h of nitrogen starvation. This loss of activity was not observed in the presence of cycloheximide alone. In a mutant yeast strain which lacks the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase, no significant decrease in amino acid transport was observed when ammonium sulfate was added to nitrogen-starved cells. A double mutant, which lacks the nicotinamide adenine dinucleotide phosphate-dependent enzyme and in addition has a derepressed level of the nicotinamide adenine dinucleotide-dependent (catabolic) glutamate dehydrogenase, shows the same sensitivity to ammonium ion as the wild-type strain. These data suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell and its subsequent incorporation into the a-amino groups of glutamate and other amino acids.
Saccharomyces cerevisiae e1278b contains an active transport system, the general amino acid permease, which has been shown to accept neutral and basic amino acids as substrates (10). This transport system has a high Vmax for most of the common amino acids in cells grown with a poor nitrogen source such as proline and is almost undetectable in cells grown on ammonium sulfate. In addition to the general permease, there are a number of specific amino acid transport systems in Saccharomyces (1, 6, 7, 11, 12). Most of these appear to be low velocity constitutive systems which are not inhibitied by ammonium ion. However, two specific transport systems, for L-proline (15) and for dicarboxylic amino acids (12), respectively, were shown to have reduced activity in the presence of ammonium ion. Growth of S. cerevisiae e1278b on ammonium ion results in the repression of a number of enzymes involved in nitrogen catabolism (3-5, 8, 16) as well as inhibition of the general amino acid permease (9). This repression is relieved in mutant strains which lack the nicotinamide adenine dinucleotide phosphate (NADP)dependent glutamate dehydrogenase (3-5, 8, 16). In addition, the general amino acid permease in these mutants is insensitive to ammonium ion inhibition (9). One specific system
that has been extensively studied is nitrogen catabolite repression of arginase in Saccharomyces. Dubois et al. have proposed that the NADP-dependent glutamate dehydrogenase molecule acts directly in the repression of arginase synthesis when the cellular levels of both ammonium ion and a-ketoglutarate are high (5). According to this proposal, repression by ammonia is not a consequence of the catalytic activity of glutamate dehydrogenase, i.e., its ability to catalyze the formation of glutamic acid, but rather results from some regulatory action of the protein molecule, possibly at the site of messenger ribonucleic acid synthesis. Moreover, since ammonium ion inhibition of the general amino acid permease is relieved concomitant with nitrogen catabolite repression, it has been suggested that the NADPdependent glutamate dehydrogenase may also serve in a direct regulatory capacity with respect to ammonium ion inhibition of amino acid transport systems (9). A number of questions concerning the mechanism of ammonia inhibition of amino acid transport remain unanswered. In the first place, there appears to be some uncertainty about the time course of this effect. Secondly, it is presently unclear whether ammonia is an external inhibitor or must be transported into the cell to
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exert its effect. Furthermore, the available data adjusted to an optical density of 0.5 at 660 nm (using do not allow one to distinguish whether ammo- a cuvette with a 10-mm light path) initially contained nium ion per se or its metabolic products are 0.17 mg of cells (dry weight) per ml. During 4 h of inhibitory and do not answer the related ques- incubation at 23 C in the glucose-phosphate transport the dry weight increased 30%. All activities tion of whether the NADP-dependent gluta- medium, are with respect to the dry weight of the reported mate dehydrogenase plays a catalytic or regula- cellular material. tory role in mediating this inhibitory effect. The Determination of radioactivity. The scintillation studies reported in the present communication fluid for determination of 'H-labeled amino acids were designed to investigate some of these contained 9.9 g of 2,5-diphenyloxazole, 609 mg of questions. 1-4-bis-(5-phenyloxazole)benzene, 3,000 ml of toluMATERIALS AND METHODS Organisms. The haploid yeast strains e1278b, 4324c(gdhA-), and 12759a(gdhA,-, gdhCR), which are isogenic except for the mutations shown (8), were obtained from M. Grenson. The nomenclature used by Grenson (8) has been retained for designating these mutant strains. Growth conditions. The standard chemically defined medium contained, per liter: 20 g of -glucose, 2 g of yeast nitrogen base (Difco no. 033-15-9, without amino acids and ammonium sulfate), and 1.32 g of ammonium sulfate. Yeast cultures were grown at 23 C with slow rotary shaking at 150 rpm from a 1% inoculum. The cells were harvested by centrifugation after approximately 16 h of growth. (At the time of harvesting the optical density of the cell suspension at 660 nm was 1.5 to 1.8, using cuvettes with a 10-mm light path.) The harvested cellular material was used immediately for the assay of amino acid transport activity. The complex (PDY) medium contained, per liter: 20 g of peptone (Difco), 20 g of D-glucose and 10 g of yeast extract (Difco). Conditions of growth were the same as those used with the chemically defined medium except that cells were harvested after approximately 12 h of growth. Assay of amino acid transport. The radioactive assay system is analogous to that described by Grenson et al. (10). Cells were suspended to 0.5 OD.., in medium containing 2% D-glucose and 20 mM potassium phosphate buffer, pH 6.2, and incubated at 23 C with slow shaking (150 rpm). At appropriate times, 4.5-ml samples were removed and mixed with 0.5 ml of 1 mM L- [9H ]alanine, L- ['H lasparagine, L- [SH Ilysine, or L- ['H ]proline (specific activity 1.0 x 103 counts/min per nmol). Samples (1.0 ml) of this mixture were removed at 30-s intervals for 2 min, poured onto membrane filters (Gelman filters, 25 mm diameter, 0.45 gm pore size), and immediately washed with 10 ml of ice cold water. The washed filters were placed in vials containing 5 ml of liquid scintillation counting fluid. Under the standard assay conditions, the rate of amino acid transport was linear with time for at least 2 min. A similar system was used for the assay of L-aspartic acid transport except that 4.5-ml samples of the cell suspension were removed, concentrated on a membrane filter, suspended in medium containing 2% D-glucose and 20 mM potassium phosphate buffer, pH 4.3, and incubated at 23 C for 5 min before being mixed with 0.5 ml of 1 mM L- [3H ]aspartic acid. Dry weight determination. A yeast suspension o
ene, and 390 ml of Bio-Solve BBS-3 (obtained from Beckman Instruments, Inc.). Radioactivity was measured on a Beckman LS100 liquid scintillation counter.
RESULTS Increase in the rate of amino acid transport in response to nitrogen starvation. The rate of transport of five L-labeled amino acids was initially low in cells which had been grown on minimal ammonia medium (Fig. 1). Transport activity increased markedly during nitrogen starvation, approaching a maximum after 3 to 5 h of incubation in the glucose phosphate assay medium. The increase in transport activity could be prevented by the addition of ammonium sulfate or cycloheximide at the beginning of the incubation (Fig. 2). Yeast cells which were grown on the complex (PDY) medium (Fig. 2) before suspension in the assay medium also had a very low initial activity but reached a maximum somewhat sooner than did ammonia-grown cells. For this reason, cells grown on PDY medium were used in most of the subsequent experiments. Decrease in transport activity after addition of ammonium ion. When ammonium sulfate was added to a cell suspension of E1278b which had been incubated for 3 h in the assay medium, there was initially very little detectable inhibition of transport activity for the five amino acids tested (Fig. 3a). However, a timedependent decrease in amino acid transport activity was observed. When mutant strain 4324c, which lacks the NADP-dependent glutamate dehydrogenase, was subjected to the same treatment, transport activity declined somewhat (10 to 30%) for the first 40 min, but then recovered to an activity level slightly higher than that initially observed (Fig. 3b). In strain 12759a, which is derepressed for the synthesis of the NAD-dependent glutamate dehydrogenase and lacks the NADP-dependent glutamate dehydrogenase, the sensitivity to ammonia inhibition was restored to that observed in the wildtype strain (Fig. 3c). The inhibition of transport activity by ammonium ion is not significantly altered when
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Hours FIG. 1. Effect of nitrogen starvation on L-labeled amino acid transport by S. cerevisiae E1278b. Cells grown on minimal ammonia medium were suspended in glucose-phosphate medium. At the times indicated, 4.5-ml samples were removed and assayed for transport activity with tritiated L-aspartate (0), L-asparagine (O), L-alanine (0), L-lysine (A) and L-proline (0).
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tions of assay and was restored in the double mutant strain. A more extensive survey of the effects of preloading with nitrogen compounds was conducted as shown in Table 1. It can be seen that all three strains gave a similar response to amino acids but that the inhibitory effects of ammonium sulfate, and of allantoin or urea (which must be degraded to ammonium ion before entering the amino acid pool), were lost in the (NADP) glutamate dehydrogenaseless strain and regained in the double mutant.
DISCUSSION The data contained in this report suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell, its subsequent incorporation into the a-amino group of glutamic acid and other a-amino acids and in the resultant rise in the level of the amino acid pool. Our results also indicate that, in contrast to the model suggested for ammonia catabolite repression (5), the NADP-dependent glutamate dehydrogenase functions in the inhibition of amino acid transport by virtue of its catalytic capacity for incorporating ammonium ion into glutamic
cycloheximide is added in addition to ammonia sulfate nor does cycloheximide alone significantly inhibit the initial rate of transport during the duration of this experiment (Fig. 4). When cells which had been incubated with ammonium sulfate were collected on a membrane filter and resuspended in ammonia-free medium, there was a slow, time-dependent recovery of transport activity. Comparison of ammonia inhibition with transinhibition by amino acids. It has been reported that the general amino acid permease of Saccharomyces is inhibited in cells which have been preloaded with L- or D-labeled amino acids (14). To determine whether a relationship exists between this type of transinhibition by amino acids and the inhibition observed in the presence of ammonium ion the following experiment was conducted. Cell suspensions were in30 60 90 120 150 180 210 cubated for 3 h in the assay medium and then minutes ammonium sulfate or amino acids were added. FiG. 2. Effect of growth medium, cycloheximide, or The cells were collected by filtration at the times indicated, suspended in fresh nitrogen- ammonium sulfate on L-alanine transport. Cells of free assay medium, and assayed for transport e1278b grown on minimal ammonia medium were suspended in glucose-phosphate medium with the activity. Transinhibition by amino acids fol- following nothing (0), 10 mM (NH4)SO4 lowed a similar time course in the wild type and (0), 10 jAgadditions: per ml (A). Cells of both mutant strains tested (Fig. 5a,b,c), E1278b grownof oncycloheximide complex PDY medium were suswhereas ammonia inhibition was not detectable pended in glucose-phosphate medium (0). At the in the strain lacking the NADP-dependgnt times indicated, 4.5-mi samples were removed and glutamate dehydrogenase under these condi- assayed for L-alanine transport activity.
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ase does not serve as a regulatory element for amino acid transport. However, one must raise the alternative explanation that the NADdependent enzyme can substitute as a regulatory element in the absence of the NADPdependent enzyme. Although our data do not eliminate this possibility, it seems somewhat E unlikely in light of the dissimilar nature of the c20 two enzymes (2). The studies on transinhibition support the conclusion that inhibition by ammonium ion is contingent on its incorporation into amino acids. The inhibitory effect of amino acids is virtually unchanged in the mutant strains, whereas the effect of ammonium ion and those compounds which are metabolized via ammonium ion are lost in the (gdhAr-) mutant and 30 60 90 120 180 210 regained in the (gdhA1-, gdhCR) mutant. The minutes time dependence of inhibition subsequent to the FIG. 4. Decrease in L-alanine transport activity in addition of ammonium ion and the slow reversal e1278b after the addition of ammonium sulfate; effect of this effect after resuspension of the cells in of cycloheximide and removal of external ammonium nitrogen-free medium are also consistent with sulfate. Conditions of assay were identical to those the proposal. given under 3a. Additions were as follows: nothing (0), The data presented in this report do not 10 mM (NH4JSO, (A), 10 lAg of cycloheximide per ml (0), 10 mM (NH)SO, and 10 ug of cyclohex- elucidate the mechanism(s) by which the transimide per ml (-). At 60 and 90 min, portions of the port of amino acids is inhibited. Further work is ammonia-containing cell suspensions (0) were filtered needed to determine whether the inhibition is the result of a direct reversible interaction of the on membrane filters and the cells were suspended in the same volume of fresh nitrogen-free medium. internal amino acid pool with the transport At the times indicated, samples (4.5 ml) were assayed systems or whether irreversible alterations in for L-alanine transport activity. these systems occur when they are in the inhibited state. The relatively slow increase in transport activity during nitrogen starvation acid and not as a regulatory element. The data suggests that some de novo protein synthesis summarized below are consistant with this may be required for recovery of transport activproposal. ity. Since the usual techniques of inhibiting The critical experimental finding is that ammonia inhibition which is lost in mutant strain 1. Inhibition of L-alanine transport in cells 4324c(gdhAc) lacking the NADP-dependent TABLE preloaded with nitrogen compounds glutamate dehydrogenase is restored in strain 12759a(gdhA,-, gdhCR). Although the latter Initial transport rate strain is still missing the anabolic enzyme it (nmollmin per mg of cells) Nitrogen compound testeda now contains derepressed levels of the NADf 1278b 4324c 12759a dependent glutamate dehydrogenase. As evidenced by the nearly normal growth rate of Control 35 36 30 strain 12659a in ammonium sulfate medium (8) L-alanine 0.4 1.4 0.5 the derepression of the NAD-dependent enzyme L-asparagine 1.6 1.6 0.5 allows it to replace the catalytic anabolic func- L-lysine 8.2 11 5.0 tion (glutamate synthesis) of the NADP- L-histidine 0.5 2.3 0.8 1.2 2.3 2.0 dependent enzyme. Since this double mutant L-valine 1.9 2.5 0.6 still lacks the structural gene for glutamate L-leucine 0.5 45 5 dehydrogenase (NADP) one would expect it to Ammonium sulfate 16 25 50 be insensitive to ammonium ion if the NADP- Urea 14 40 17.5 dependent enzyme were functioning as a regula- Allantoin a tory element. Since sensitivity to ammonium Conditions of assay were identical to those given ion is restored, the simplest conclusion is that in Fig. 5 except that the cells were incubated with the the NADP-dependent glutamate dehydrogen- inhibitors for 2 h before being assayed.
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J. BACTERIOL.
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Minutes FIG. 5. Inhibition of L-alanine transport in cells preloaded with L-labeled amino acids and ammonium sulfate. (a) Conditions of assay were identical to those given under 3a. After 3 h of incubation, inhibitors were added to a final concentration of 10 mM as follows: (NHJ2SO, (@), L-lysine (A), L-asparagine (0), L-alanine (0), L-histidine (U). At the times indicated, 4.5-ml samples were removed and the cells were collected on membrane filters and washed with 10-mi samples of assay medium at 23 C. The ceUs were then suspended to 4.5 ml and assayed for L-alanine transport activity after 5 min. (b) Conditions were identical to 5a except that strain 4324c(gdhA,-) was assayed. (c) Conditions were identical to 5a except that strain 12759a(gdhA,-, gdhCr) was assayed.
protein synthesis may simultaneously freeze the amino acid pools at a high level, it will require a careful study of the time dependency of amino acid pool depletion and resynthesis in comparison with changes in transport activities to answer these questions. ACKNOWLEDGMENT This work was supported by American Cancer Society Research grant no. BC-126. LITERATURE CITED 1. Crabeel. M., and M. Grenson. 1970. Regulation of histidine tiptake by specific feedback inhibition of two histidine perinieases in Saccharomyces cerevisiae. Eur.
J. Biochem. 14:197-204. 2. Doherty, D. 1970. L-glutamate dehydrogenases (yeast), p. 850-856. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 17. Academic Press Inc.,
New York. 3. Dubois, E. L., and M. Grenson. 1974. Absence of involvement of glutamine synthetase and of NAD-linked glutamate dehydrogenase in the nitrogen catabolite repression of arginase and other enzymes in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 60:150-157. 4. Dubois, E., M. Grenson, and J. M. Wiame. 1973. Release of the "ammonia effect" on three catabolic enzymes by NADP-specific glutamate dehydrogenaseless mutations in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 50:967-972. 5. Dubois, E., M. Grenson, and J. Wiame. 1974. The
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6.
7.
8.
9.
10.
participation of the anabolic glutamate dehydrogenase in the nitrogen catabolite repression of arginase in Saccharomyces cerevisiae. Eur. J. Biochem. 48:603-616. Gits, J., and M. Grenson. 1967. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. Im. Evidence for a specific methionine-transporting system. Biochim. Biophys. Acta 135:507-516. Grenson, M. 1966. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. H. Evidence for a specific lysine-transporting system. Biochim. Biophys. Acta 127:339-346. Grenson, M., E. Dubois, M. Piotrowska, R. Drillien, and M. Aigle. 1974. Ammonia assimilation in Saccharomyces cerevisiae as mediated by the two glutamate dehydrogenases. Mol. Gen. Genet. 128:73-85. Grenson, M., and C. Hou. 1972. Ammonia inhibition of the general amino acid permease and its suppression in NADPH-specific glutamate dehydrogenaseless mutants of Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 48:749-756. Grenson, M., C. Hou, and M. Crabeel. 1970. Multiplicity of amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease. J.
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Bacteriol. 103:770-777. 11. Grenson, M., M. Mousset, J. M. Wiame, and J. Bechet. 1966. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. I. Evidence for a specific arginine-transporting system. Biochim. Biophys. Acta 127:325-338. 12. Joiris, C. R., and M. Grenson. 1969. Specificite et regulation d'une per m6ase des acides amines dicarboxyliques chez Saccharomyces cerevisiae. Arch. Int. Physiol. Biochem. 77:154-156. 13. Roon, R., H. L. Even, P. Dunlop, and F. L. Larimore, 1975. Methylamine and ammonia transport in Saccharomyces cerevisiae. J. Bacteriol. 122:502-509. 14. Rytka, J. 1975. Positive selection of general amino acid permease mutants in Saccharomyces cerevisiae. J. Bacteriol. 121:562-570. 15. Schwencke, J., and N. Magafia-Schwencke. 1969. Derepression of a proline transport system in Saccharomyces chevalieri by nitrogen starvation. Biochem. Biophys. Acta 173:302-312. 16. Wiame, J. M. 1973. In Metabolism and cellular processes, part II, p. 307-330. In H. Suomalainen and C. Waller ed.), Proc. Third Int. Specialized Symp. on Yeast,
Helsinki.
Vol. 124, Ng. 1 Printed in U.S.A.
Inhibition of Amino Acid Transport by Ammonium Ion in Saccharomyces cerevisiae ROBERT J. ROON,* FRED LARIMORE, AND JANE S. LEYY
Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 55455 Received for publication 25 June 1975
The rate of transport of L-amino acids by Saccharomyces cerevisiae e1278b increased with time in response to nitrogen starvation. This increase could be prevented by the addition of ammonium sulfate or cycloheximide. A slow time-dependent loss of transport activity was observed when ammonium sulfate (or ammonium sulfate plus cycloheximide) was added to cells after 3 h of nitrogen starvation. This loss of activity was not observed in the presence of cycloheximide alone. In a mutant yeast strain which lacks the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase, no significant decrease in amino acid transport was observed when ammonium sulfate was added to nitrogen-starved cells. A double mutant, which lacks the nicotinamide adenine dinucleotide phosphate-dependent enzyme and in addition has a derepressed level of the nicotinamide adenine dinucleotide-dependent (catabolic) glutamate dehydrogenase, shows the same sensitivity to ammonium ion as the wild-type strain. These data suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell and its subsequent incorporation into the a-amino groups of glutamate and other amino acids.
Saccharomyces cerevisiae e1278b contains an active transport system, the general amino acid permease, which has been shown to accept neutral and basic amino acids as substrates (10). This transport system has a high Vmax for most of the common amino acids in cells grown with a poor nitrogen source such as proline and is almost undetectable in cells grown on ammonium sulfate. In addition to the general permease, there are a number of specific amino acid transport systems in Saccharomyces (1, 6, 7, 11, 12). Most of these appear to be low velocity constitutive systems which are not inhibitied by ammonium ion. However, two specific transport systems, for L-proline (15) and for dicarboxylic amino acids (12), respectively, were shown to have reduced activity in the presence of ammonium ion. Growth of S. cerevisiae e1278b on ammonium ion results in the repression of a number of enzymes involved in nitrogen catabolism (3-5, 8, 16) as well as inhibition of the general amino acid permease (9). This repression is relieved in mutant strains which lack the nicotinamide adenine dinucleotide phosphate (NADP)dependent glutamate dehydrogenase (3-5, 8, 16). In addition, the general amino acid permease in these mutants is insensitive to ammonium ion inhibition (9). One specific system
that has been extensively studied is nitrogen catabolite repression of arginase in Saccharomyces. Dubois et al. have proposed that the NADP-dependent glutamate dehydrogenase molecule acts directly in the repression of arginase synthesis when the cellular levels of both ammonium ion and a-ketoglutarate are high (5). According to this proposal, repression by ammonia is not a consequence of the catalytic activity of glutamate dehydrogenase, i.e., its ability to catalyze the formation of glutamic acid, but rather results from some regulatory action of the protein molecule, possibly at the site of messenger ribonucleic acid synthesis. Moreover, since ammonium ion inhibition of the general amino acid permease is relieved concomitant with nitrogen catabolite repression, it has been suggested that the NADPdependent glutamate dehydrogenase may also serve in a direct regulatory capacity with respect to ammonium ion inhibition of amino acid transport systems (9). A number of questions concerning the mechanism of ammonia inhibition of amino acid transport remain unanswered. In the first place, there appears to be some uncertainty about the time course of this effect. Secondly, it is presently unclear whether ammonia is an external inhibitor or must be transported into the cell to
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exert its effect. Furthermore, the available data adjusted to an optical density of 0.5 at 660 nm (using do not allow one to distinguish whether ammo- a cuvette with a 10-mm light path) initially contained nium ion per se or its metabolic products are 0.17 mg of cells (dry weight) per ml. During 4 h of inhibitory and do not answer the related ques- incubation at 23 C in the glucose-phosphate transport the dry weight increased 30%. All activities tion of whether the NADP-dependent gluta- medium, are with respect to the dry weight of the reported mate dehydrogenase plays a catalytic or regula- cellular material. tory role in mediating this inhibitory effect. The Determination of radioactivity. The scintillation studies reported in the present communication fluid for determination of 'H-labeled amino acids were designed to investigate some of these contained 9.9 g of 2,5-diphenyloxazole, 609 mg of questions. 1-4-bis-(5-phenyloxazole)benzene, 3,000 ml of toluMATERIALS AND METHODS Organisms. The haploid yeast strains e1278b, 4324c(gdhA-), and 12759a(gdhA,-, gdhCR), which are isogenic except for the mutations shown (8), were obtained from M. Grenson. The nomenclature used by Grenson (8) has been retained for designating these mutant strains. Growth conditions. The standard chemically defined medium contained, per liter: 20 g of -glucose, 2 g of yeast nitrogen base (Difco no. 033-15-9, without amino acids and ammonium sulfate), and 1.32 g of ammonium sulfate. Yeast cultures were grown at 23 C with slow rotary shaking at 150 rpm from a 1% inoculum. The cells were harvested by centrifugation after approximately 16 h of growth. (At the time of harvesting the optical density of the cell suspension at 660 nm was 1.5 to 1.8, using cuvettes with a 10-mm light path.) The harvested cellular material was used immediately for the assay of amino acid transport activity. The complex (PDY) medium contained, per liter: 20 g of peptone (Difco), 20 g of D-glucose and 10 g of yeast extract (Difco). Conditions of growth were the same as those used with the chemically defined medium except that cells were harvested after approximately 12 h of growth. Assay of amino acid transport. The radioactive assay system is analogous to that described by Grenson et al. (10). Cells were suspended to 0.5 OD.., in medium containing 2% D-glucose and 20 mM potassium phosphate buffer, pH 6.2, and incubated at 23 C with slow shaking (150 rpm). At appropriate times, 4.5-ml samples were removed and mixed with 0.5 ml of 1 mM L- [9H ]alanine, L- ['H lasparagine, L- [SH Ilysine, or L- ['H ]proline (specific activity 1.0 x 103 counts/min per nmol). Samples (1.0 ml) of this mixture were removed at 30-s intervals for 2 min, poured onto membrane filters (Gelman filters, 25 mm diameter, 0.45 gm pore size), and immediately washed with 10 ml of ice cold water. The washed filters were placed in vials containing 5 ml of liquid scintillation counting fluid. Under the standard assay conditions, the rate of amino acid transport was linear with time for at least 2 min. A similar system was used for the assay of L-aspartic acid transport except that 4.5-ml samples of the cell suspension were removed, concentrated on a membrane filter, suspended in medium containing 2% D-glucose and 20 mM potassium phosphate buffer, pH 4.3, and incubated at 23 C for 5 min before being mixed with 0.5 ml of 1 mM L- [3H ]aspartic acid. Dry weight determination. A yeast suspension o
ene, and 390 ml of Bio-Solve BBS-3 (obtained from Beckman Instruments, Inc.). Radioactivity was measured on a Beckman LS100 liquid scintillation counter.
RESULTS Increase in the rate of amino acid transport in response to nitrogen starvation. The rate of transport of five L-labeled amino acids was initially low in cells which had been grown on minimal ammonia medium (Fig. 1). Transport activity increased markedly during nitrogen starvation, approaching a maximum after 3 to 5 h of incubation in the glucose phosphate assay medium. The increase in transport activity could be prevented by the addition of ammonium sulfate or cycloheximide at the beginning of the incubation (Fig. 2). Yeast cells which were grown on the complex (PDY) medium (Fig. 2) before suspension in the assay medium also had a very low initial activity but reached a maximum somewhat sooner than did ammonia-grown cells. For this reason, cells grown on PDY medium were used in most of the subsequent experiments. Decrease in transport activity after addition of ammonium ion. When ammonium sulfate was added to a cell suspension of E1278b which had been incubated for 3 h in the assay medium, there was initially very little detectable inhibition of transport activity for the five amino acids tested (Fig. 3a). However, a timedependent decrease in amino acid transport activity was observed. When mutant strain 4324c, which lacks the NADP-dependent glutamate dehydrogenase, was subjected to the same treatment, transport activity declined somewhat (10 to 30%) for the first 40 min, but then recovered to an activity level slightly higher than that initially observed (Fig. 3b). In strain 12759a, which is derepressed for the synthesis of the NAD-dependent glutamate dehydrogenase and lacks the NADP-dependent glutamate dehydrogenase, the sensitivity to ammonia inhibition was restored to that observed in the wildtype strain (Fig. 3c). The inhibition of transport activity by ammonium ion is not significantly altered when
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Hours FIG. 1. Effect of nitrogen starvation on L-labeled amino acid transport by S. cerevisiae E1278b. Cells grown on minimal ammonia medium were suspended in glucose-phosphate medium. At the times indicated, 4.5-ml samples were removed and assayed for transport activity with tritiated L-aspartate (0), L-asparagine (O), L-alanine (0), L-lysine (A) and L-proline (0).
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tions of assay and was restored in the double mutant strain. A more extensive survey of the effects of preloading with nitrogen compounds was conducted as shown in Table 1. It can be seen that all three strains gave a similar response to amino acids but that the inhibitory effects of ammonium sulfate, and of allantoin or urea (which must be degraded to ammonium ion before entering the amino acid pool), were lost in the (NADP) glutamate dehydrogenaseless strain and regained in the double mutant.
DISCUSSION The data contained in this report suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell, its subsequent incorporation into the a-amino group of glutamic acid and other a-amino acids and in the resultant rise in the level of the amino acid pool. Our results also indicate that, in contrast to the model suggested for ammonia catabolite repression (5), the NADP-dependent glutamate dehydrogenase functions in the inhibition of amino acid transport by virtue of its catalytic capacity for incorporating ammonium ion into glutamic
cycloheximide is added in addition to ammonia sulfate nor does cycloheximide alone significantly inhibit the initial rate of transport during the duration of this experiment (Fig. 4). When cells which had been incubated with ammonium sulfate were collected on a membrane filter and resuspended in ammonia-free medium, there was a slow, time-dependent recovery of transport activity. Comparison of ammonia inhibition with transinhibition by amino acids. It has been reported that the general amino acid permease of Saccharomyces is inhibited in cells which have been preloaded with L- or D-labeled amino acids (14). To determine whether a relationship exists between this type of transinhibition by amino acids and the inhibition observed in the presence of ammonium ion the following experiment was conducted. Cell suspensions were in30 60 90 120 150 180 210 cubated for 3 h in the assay medium and then minutes ammonium sulfate or amino acids were added. FiG. 2. Effect of growth medium, cycloheximide, or The cells were collected by filtration at the times indicated, suspended in fresh nitrogen- ammonium sulfate on L-alanine transport. Cells of free assay medium, and assayed for transport e1278b grown on minimal ammonia medium were suspended in glucose-phosphate medium with the activity. Transinhibition by amino acids fol- following nothing (0), 10 mM (NH4)SO4 lowed a similar time course in the wild type and (0), 10 jAgadditions: per ml (A). Cells of both mutant strains tested (Fig. 5a,b,c), E1278b grownof oncycloheximide complex PDY medium were suswhereas ammonia inhibition was not detectable pended in glucose-phosphate medium (0). At the in the strain lacking the NADP-dependgnt times indicated, 4.5-mi samples were removed and glutamate dehydrogenase under these condi- assayed for L-alanine transport activity.
328
ROON, LARIMORE, AND LEVY
J. BACTERIOL.
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INHIBITION OF AMINO ACID TRANSPORT
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329
ase does not serve as a regulatory element for amino acid transport. However, one must raise the alternative explanation that the NADdependent enzyme can substitute as a regulatory element in the absence of the NADPdependent enzyme. Although our data do not eliminate this possibility, it seems somewhat E unlikely in light of the dissimilar nature of the c20 two enzymes (2). The studies on transinhibition support the conclusion that inhibition by ammonium ion is contingent on its incorporation into amino acids. The inhibitory effect of amino acids is virtually unchanged in the mutant strains, whereas the effect of ammonium ion and those compounds which are metabolized via ammonium ion are lost in the (gdhAr-) mutant and 30 60 90 120 180 210 regained in the (gdhA1-, gdhCR) mutant. The minutes time dependence of inhibition subsequent to the FIG. 4. Decrease in L-alanine transport activity in addition of ammonium ion and the slow reversal e1278b after the addition of ammonium sulfate; effect of this effect after resuspension of the cells in of cycloheximide and removal of external ammonium nitrogen-free medium are also consistent with sulfate. Conditions of assay were identical to those the proposal. given under 3a. Additions were as follows: nothing (0), The data presented in this report do not 10 mM (NH4JSO, (A), 10 lAg of cycloheximide per ml (0), 10 mM (NH)SO, and 10 ug of cyclohex- elucidate the mechanism(s) by which the transimide per ml (-). At 60 and 90 min, portions of the port of amino acids is inhibited. Further work is ammonia-containing cell suspensions (0) were filtered needed to determine whether the inhibition is the result of a direct reversible interaction of the on membrane filters and the cells were suspended in the same volume of fresh nitrogen-free medium. internal amino acid pool with the transport At the times indicated, samples (4.5 ml) were assayed systems or whether irreversible alterations in for L-alanine transport activity. these systems occur when they are in the inhibited state. The relatively slow increase in transport activity during nitrogen starvation acid and not as a regulatory element. The data suggests that some de novo protein synthesis summarized below are consistant with this may be required for recovery of transport activproposal. ity. Since the usual techniques of inhibiting The critical experimental finding is that ammonia inhibition which is lost in mutant strain 1. Inhibition of L-alanine transport in cells 4324c(gdhAc) lacking the NADP-dependent TABLE preloaded with nitrogen compounds glutamate dehydrogenase is restored in strain 12759a(gdhA,-, gdhCR). Although the latter Initial transport rate strain is still missing the anabolic enzyme it (nmollmin per mg of cells) Nitrogen compound testeda now contains derepressed levels of the NADf 1278b 4324c 12759a dependent glutamate dehydrogenase. As evidenced by the nearly normal growth rate of Control 35 36 30 strain 12659a in ammonium sulfate medium (8) L-alanine 0.4 1.4 0.5 the derepression of the NAD-dependent enzyme L-asparagine 1.6 1.6 0.5 allows it to replace the catalytic anabolic func- L-lysine 8.2 11 5.0 tion (glutamate synthesis) of the NADP- L-histidine 0.5 2.3 0.8 1.2 2.3 2.0 dependent enzyme. Since this double mutant L-valine 1.9 2.5 0.6 still lacks the structural gene for glutamate L-leucine 0.5 45 5 dehydrogenase (NADP) one would expect it to Ammonium sulfate 16 25 50 be insensitive to ammonium ion if the NADP- Urea 14 40 17.5 dependent enzyme were functioning as a regula- Allantoin a tory element. Since sensitivity to ammonium Conditions of assay were identical to those given ion is restored, the simplest conclusion is that in Fig. 5 except that the cells were incubated with the the NADP-dependent glutamate dehydrogen- inhibitors for 2 h before being assayed.
C-)
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330
ROON, LARIMORE, AND LEVY
J. BACTERIOL.
a)
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Minutes FIG. 5. Inhibition of L-alanine transport in cells preloaded with L-labeled amino acids and ammonium sulfate. (a) Conditions of assay were identical to those given under 3a. After 3 h of incubation, inhibitors were added to a final concentration of 10 mM as follows: (NHJ2SO, (@), L-lysine (A), L-asparagine (0), L-alanine (0), L-histidine (U). At the times indicated, 4.5-ml samples were removed and the cells were collected on membrane filters and washed with 10-mi samples of assay medium at 23 C. The ceUs were then suspended to 4.5 ml and assayed for L-alanine transport activity after 5 min. (b) Conditions were identical to 5a except that strain 4324c(gdhA,-) was assayed. (c) Conditions were identical to 5a except that strain 12759a(gdhA,-, gdhCr) was assayed.
protein synthesis may simultaneously freeze the amino acid pools at a high level, it will require a careful study of the time dependency of amino acid pool depletion and resynthesis in comparison with changes in transport activities to answer these questions. ACKNOWLEDGMENT This work was supported by American Cancer Society Research grant no. BC-126. LITERATURE CITED 1. Crabeel. M., and M. Grenson. 1970. Regulation of histidine tiptake by specific feedback inhibition of two histidine perinieases in Saccharomyces cerevisiae. Eur.
J. Biochem. 14:197-204. 2. Doherty, D. 1970. L-glutamate dehydrogenases (yeast), p. 850-856. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 17. Academic Press Inc.,
New York. 3. Dubois, E. L., and M. Grenson. 1974. Absence of involvement of glutamine synthetase and of NAD-linked glutamate dehydrogenase in the nitrogen catabolite repression of arginase and other enzymes in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 60:150-157. 4. Dubois, E., M. Grenson, and J. M. Wiame. 1973. Release of the "ammonia effect" on three catabolic enzymes by NADP-specific glutamate dehydrogenaseless mutations in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 50:967-972. 5. Dubois, E., M. Grenson, and J. Wiame. 1974. The
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6.
7.
8.
9.
10.
participation of the anabolic glutamate dehydrogenase in the nitrogen catabolite repression of arginase in Saccharomyces cerevisiae. Eur. J. Biochem. 48:603-616. Gits, J., and M. Grenson. 1967. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. Im. Evidence for a specific methionine-transporting system. Biochim. Biophys. Acta 135:507-516. Grenson, M. 1966. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. H. Evidence for a specific lysine-transporting system. Biochim. Biophys. Acta 127:339-346. Grenson, M., E. Dubois, M. Piotrowska, R. Drillien, and M. Aigle. 1974. Ammonia assimilation in Saccharomyces cerevisiae as mediated by the two glutamate dehydrogenases. Mol. Gen. Genet. 128:73-85. Grenson, M., and C. Hou. 1972. Ammonia inhibition of the general amino acid permease and its suppression in NADPH-specific glutamate dehydrogenaseless mutants of Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 48:749-756. Grenson, M., C. Hou, and M. Crabeel. 1970. Multiplicity of amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease. J.
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Bacteriol. 103:770-777. 11. Grenson, M., M. Mousset, J. M. Wiame, and J. Bechet. 1966. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. I. Evidence for a specific arginine-transporting system. Biochim. Biophys. Acta 127:325-338. 12. Joiris, C. R., and M. Grenson. 1969. Specificite et regulation d'une per m6ase des acides amines dicarboxyliques chez Saccharomyces cerevisiae. Arch. Int. Physiol. Biochem. 77:154-156. 13. Roon, R., H. L. Even, P. Dunlop, and F. L. Larimore, 1975. Methylamine and ammonia transport in Saccharomyces cerevisiae. J. Bacteriol. 122:502-509. 14. Rytka, J. 1975. Positive selection of general amino acid permease mutants in Saccharomyces cerevisiae. J. Bacteriol. 121:562-570. 15. Schwencke, J., and N. Magafia-Schwencke. 1969. Derepression of a proline transport system in Saccharomyces chevalieri by nitrogen starvation. Biochem. Biophys. Acta 173:302-312. 16. Wiame, J. M. 1973. In Metabolism and cellular processes, part II, p. 307-330. In H. Suomalainen and C. Waller ed.), Proc. Third Int. Specialized Symp. on Yeast,
Helsinki.
