Vol. 121, No. 2 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Feb. 1975, p. 562-570 Copyright 0 1975 American Society for Microbiology

Positive Selection of General Amino Acid Permease Mutants in Saccharomyces cerevisiae JOANNA RYTKA Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-532, Warszawa, Poland

Received for publication 15 November 1974

It was found that D-stereoisomers of natural amino acids inhibit the growth of Saccharomyces cerevisiae cells. Kinetic and genetic evidence showed that D-amino acids enter the cell by the general amino acid permease. Two classes of S. cerevisiae mutants resistant to D-amino acids were isolated. One class of mutants appeared to be defective in the general amino acid permease specified by the gene gap. In the second class, the activity of general amino acid permease was affected by ammonium ions. Mutants of the second class were isolated in a yeast strain with the general amino acid permease insensitive to the presence of ammonium ions in culture media. The mutation affecting the permease, amc, occurred in a locus unlinked to gap. A number of specific amino acid permeases have been found in the yeast Saccharomyces cerevisiae (3, 4, 5, 9). Also, the transport system common to most of the L-amino acids has been described (8, 10, 20, 21). The existence of at least two transport systems for some amino acids made the isolation of permeaseless mutants difficult. In this paper, the active transport of Dstereoisomers of natural amino acids by S. cerevisiae cells is described. D-Amino acids enter the cell by a single transport system shared with L-amino acids. D-Amino acids accumulated within yeast cells are toxic and stop the growth of S. cerevisiae. The elimination of D-amino acid transport prevents the inhibitory effect of these compounds, making it possible to isolate resistant mutants. The mutants obtained were affected in the general amino acid permease; therefore it was possible by a single mutation to eliminate one of the amino acid transport systems. Since very little is known about the regulation of amino acid uptake in yeast (2, 3, 6, 7, 8), the use of D-amino acids as genetic and biochemical probes should be a useful approach to this problem. MATERIALS AND METHODS Organism. All strains used were haploid-hetero-

thalic strains of S. cerevisiae. They are presented in Table 1.

Media and culture methods. The complete medium YPD contained 1% yeast extract (Difco), 2% peptone (Difco), and 2% glucose. The minimal salt medium Am was prepared as described by Lacroute 56:

(13). Pm medium was identical with Am medium except that ammonium sulfate was replaced by L-proline (0.5 mg/ml) as nitrogen source. Media for petri plates contained 2% agar (Difco). Cells were grown aerobically at 30 C in a rotatory shaker. The growth was followed by measuring the absorbancy at 750 nm. Cell density was estimated from a dry weight-turbidity curve previously calibrated with yeast cell suspensions.

Genetic analysis. The methods described by Hawthorne and Mortimer (11) were used. Diploids were obtained by mixing strains of opposite mating types on YPD plates. After 4 to 5 h of incubation at 30 C, the zygotes were isolated by micromanipulation and purified by several single colony reisolations on YPD plates.

Measurement of amino acid uptake. The 11Clabeled amino acids were added to exponentially growing cultures. Samples of 1 ml were removed periodically for 5 min, rapidly filtered on Coli 5 membrane filters (Biomed-Poland), and washed with 20 ml of distilled water at room temperature. The filters were dried and the radioactivity was counted in a Packard scintillation counter. Chemicals. All chemicals were obtained from commercial sources. "C-labeled amino acids were obtained from Radiochemical Centre, Amersham, England, or NEN Corp., Boston, Mass. Unlabeled Lamino acids and their D-stereoisomers were obtained from Calbiochem, Luzern, Switzerland; Fluka, Buchs, Switzerland; Mann Research Laboratories, New York; or Reanal, Budapest, Hungary.

RESULTS Inhibition of growth of S. cerevisiae by D-amino acids. It has been observed that Dstereoisomers of the natural amino acids inhibit the growth of yeast S. cerevisiae. This inhibi-

VOL. 121, 1975

SELECTION OF AMINO A(CID PERMEASE MUTANTS

TABLE 1. List of strains of S. cerevisiae used Stock

Genotype

S288C A3627A XT3003A J36U

a amca amca ad,-, amca ura amc-

21278b 2512c JR101-JR109 JR111-JR116 JR211 JR212

a amc+ a gap amc+ a ad,-, gap amca ura gap amca ura amc+ a ura gap amc+

a

Origina G. R. Fink G. R. Fink G. R. Fink EMS mutagenesis of strain A3627A M. Grenson M. Grenson This study This study This study This study

EMS, Ethylmethanesulfonate.

tory effect could be tested by an auxanographic

technique. A few crystals of D-amino acid were placed on the surface of Pm plates and overlayered with 2 ml of soft agar (0.3%) containing yeast cells. After 2 days of incubation at 30 C, a clear zone of inhibition around the crystals was observed. The same ihhibitory effect occurred in liquid media. In the experiment shown in Fig. 1, strain XT3003A and Pm medium containing L-proline as sole nitrogen source were used. There was no

563

growth in the presence of 0.5 mM D-histidine, D-methionine, or D-serine for at least 20 h. A similar result (not shown) was obtained with D-phenylalanine, D-leucine, D-alanine, or Dtryptophan. Other D-amino acids were not tested. The inhibition could be observed at concentrations as low as 0.5 mM (0.2 mM for D-histidine). The addition of D-histidine to a growing culture did not stop the growth immediately: the growth continued for one-half to one cell mass doubling (Fig. 2). When a D-amino acid was added to the medium prior to inoculation, it prevented growth for 24 to 60 h, the actual time depending on the D-amino acid used. Resumption of growth occurred at rates gradually approaching the rate of a noninhibited culture. When Damino acids were compared at identical concentrations, the longest lags were produced by D-histidine. However, when fresh medium was inoculated with washed cells grown in the presence of a D-amino acid, the cells were still D-amino acid sensitive. It follows, therefore, that the growth resumption was not due to an outgrowth of spontaneous D-amino acid-resistant mutants. Moreover, resumption of growth is not due to exhaustion of the free D-amino acid since, when radioactive D-serine was added to the culture, the amount of radioactivity mea-

3Q1.0

r_ 0:3

0.1 O TIME. h FIG. 1. Effect of D-amino acids on growth of the strain XT3003A on the minimal proline medium. Cultures were incubated at 30 C with the following additions: 0, control, no additions; 0, D-histidine (0.5 mM); A, D-serine (0.5 mM); 0, D-methionine (0.5 mM).

4

8

12

TIME, h FIG. 2. Effect of the delayed addition of D-histidine to exponentially growing cultures of the strain XT3003A. D-Histidine (0.5 mM) was added in the following time intervals: 0, 0 time; 0, after 2 h of growth; A, after 4 h of growth; 0, control, no additions.

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564

sured in the culture supernatant after growth resumption was practically the same as at the beginning, and all of it could be recovered in a single chromatographic spot with an R, identical with that of free D-serine. In yeast, D-amino acids are converted to N-acetyl D-amino acids (22). Preliminary experiments suggested that the accumulation of Nacetyl D-amino acids inside the cell prevented further uptake of D-amino acids. The growth inhibitory effect of D-amino acids could not be relieved by washing the cells. When cells were preloaded for 2 h with 10 mM D-histidine, washed, and then used for inoculating fresh medium without any D-amino acid supplement, growth did not appear immediately, but only after a 6- to 10-h lag (Fig. 3). Supplementing media with an L-amino acid at the concentrations of 0.1 to 0.2 mM prevented the growth inhibitory effect of any of the D-amino acids tested. No structural relationship was observed. For example, inhibition by 0.5 mM D-methionine could be prevented by 0.1 mM L-histidine, L-serine, L-alanine, or L-tyrosine. These effects were obserx ed also on solid media. When agar meditum contained any Lamino acid, none of the amino acids tested produced a zone of inhibition. Delayed addition of an L-amino acid to a -

6

12 TIME h

culture inhibited by a D-amino acid did not produce a recovery of growth. This suggests that L-amino acids did not counteract D-amino acid toxicity but rather prevented their growth inhibitory effect, possibly at the transport level. It was noticed that some wild-type strains of S. cerevisiae differed in their sensitivity to D-amino acids on Am medium. Strain S288C and XT300A (derived from S288C) were sensitive to D-amino acids on either Am or Pm medium. On the other hand, strain 2f1278b was inhibited by D-amino acids on Pm but not on Am medium. Uptake of D-amino acids by cells. Little was known previously concerning transport of Damino acids by yeast cells. The only reports published were on uptake of D-glutamate and D-methionine (8). Therefore, a study on specificity of D-amino acid transport was undertaken. In preliminary experiments it was observed that uptake of labeled D-amino acids was slow and therefore growing cultures were used for rate determination. D-Histidine and D-serine radioactivity taken up by the cells was in the soluble fraction. No label could be detected in trichloroacetic acid-precipitable material. The time course of 0.1 mM D-["C]histidine and D- ["C Iserine uptake by strain XT3003A is shown in Fig. 4. The initial uptake rates of the D-amino acids were 15.6 and 6.0 nmol/min per mg (dry weight) of cells, respectively. The apparent affinity constants, Km, were

18

FIG. 3. Effect of preloading the cells with D-histidine on the initial growth rates. To the cells of strain XT3003A growing exponentially in Pm medium, Dhistidine was added to final concentration 10 mM. After 2 h of incubation at 30 C, the cells were washed three times with Am medium, resuspended in Am medium, and used to inoculate fresh Am medium. Symbols: 0, control, no additions; *, inoculum preloaded with D-histidine.

5

10

TIME min

FIG. 4. Time-course of .D-histidine and D-serine uptake by XT3003A strain. Symbols: 0, 0.1 mM D-histidine; A, 0.1 mM D -serine.

SELECTION OF AMINO ACID PERMEASE MUTANTS

VOL. 121, 1975

0.025 mM for D-histidine and 0.5 mM for D-serine. The initial rates of transport of 0.01 mM D-serine and D-tyrosine were measured in the presence of other, unlabeled D-amino acids present at 10-fold higher concentration. The results shown in Table 2 demonstrate that all D-amino acids tested decreased the uptake of D-serine and D-tyrosine. The strongest inhibition, 80%o, was observed with D-histidine. DArginine was the poorest inhibitor (20% inhibition of D-serine uptake and 12% inhibition of D-tyrosine uptake). The difference in the degree of inhibition probably reflects differences in the affinity of the transport system for individual D-amino acids.

In the experiments shown in Table 3, the effect of L-amino acids on uptake of D-serine by yeast cells was studied. All five structurally unrelated L-amino acids (L-arginine, L-citrulline, L-histidine, L-leucine, and L-serine) present at 10-fold higher concentration than that of D-serine inhibited its uptake by more than 80%. This result indicates that L-amino acids interfere with the uptake of a D-amino acid more efficiently than do D-amino acids (compare Table 2 and 3). Since wild-type strains S288C and Z1278b TABLE 2. Inhibition of D-serine and D-tyrosine uptake by other D-amino acidsa Additions (0.1 mM)

Initial upInitial uptake rate Inhibi- take rate Inhibiof D-tyro- tion(%) of D-serine tion ~(%) sine (0.01 MM)b (0.01 M)bM M )b

None D-Histidine D-Tryptophan D-Methionine D-Leucine D-Arginine D-Serine D-Tyrosine

1.12 0.22 0.31 0.67 0.71 0.89

80 63 40 36 20

0.70

35

0.70 0.07 0.24 0.31 0.53 0.61 0.35

90 65 56 24 12 50

Strain XT3003A grown exponentially in minimal proline medium was used. Uptake was assayed as described. To a 4.9-ml portion of the culture, 50 1sliters of 10 mM 12C D-amino acid was added. Reaction was started by addition of 50 lAliters of 1 mM

TABLE 3. Inhibition of D-serine up,take by L-amino acidsa Additions (0.1 mM) mm) (0.1

None L-Arginine L-Citrulline L-Histidine L-Leucine L-Serine

water.

bValues expressed as nanomoles milligram (dry weight) of cells.

per minute per

Initial uptake rate of D-serine (0.01 MM)b

Inhibition (%)

1.12 0.22 0.11 0.17 0.17 0.09

80 90 85 85 92

Experimental conditions as in Table 2. b Values expressed as nanomoles per minute per milligram (dry weight) of cells. Specific radioactivity 6,600 counts/min per nmol. a

differ in their sensitivity to D-amino acids depending on the nitrogen source, the effect of their growth in the presence of either ammonium sulfate or L-proline on the uptake of D-serine was examined. Results of this experiment are shown in Table 4. Cells of either strain had essentially the same initial rate of D-serine uptake when grown on L-proline as the sole nitrogen source. On ammonium sulfate, the strain S288C had the same uptake rate. Cells of the other strain, :1278b, grown in Am medium, transported D-serine at a rate reduced by more than 100-fold as compared with cells of the same strain using L-proline as nitrogen source. This result explains the observed D-amino acid resistance of :1278b in Am medium: 99% reduction of D-amino acid uptake can fully account for its conditional resistance. Transinhibition of D-amino acid uptake. The inhibition of amino acid transport by intracellular amino acid pools was demonstrated for several transport systems and is known as transinhibition (1, 3, 12, 14, 15, 16). TABLE 4. Effect of the nitrogen source on the uptake rate of D-serine by strains S288C, Z1278b, and JR211 Initial uptake rate of D[14C serine (0.1 mM)a

a

D-["4C]tyrosine (specific radioactivity 34,500 counts/ min per nmol) or D-["4C]serine (specific radioactivity 6,600 counts/min per nmol). Samples of 1.0 ml were taken every 1 min for 5 min after the addition of the labeled D-amino acid. In the control samples, the unlabeled amino acid was replaced by 50 Mliters of

565

Nitrogen source

L-Proline Ammonium sulfate

Strain S288C

Strain 1278b

Strain JR211

7.4

5.6

6.8

0.05

6.4 0.15

a The initial uptake rate of 0.1 mM D-serine was determined by standard procedure in the cells grown exponentially in Am or Pm medium. Values are expressed as nanomoles per minute per milligram (dry weight) of cells. Specific radioactivity 6,600 counts/min per nmol.

RYTKA

566

J. BACTERIOL.

Figure 5 shows the inhibition of the uptake of D-["4C]histidine and D-[14C]serine by XT3003A cells preloaded with cold D-histidine. The initial uptake of D-serine was almost completely inhib0~~~~~ ited. Transport of D-histidine was inhibited by about 90% as compared with cells preincubated I 20 under the same conditions but without D-histidine preloading. As shown in Fig. 6 after 30 min of preloading with unlabeled D-methionine, the , 15 uptake of D- ["C ]tyrosine was noticeably decreased, and after 1 h, inhibition of 50% of uptake was reached. The data presented in E 10 Table 5 and 6 show that preloading cells with E D-histidine, D-serine, or D-tyrosine lowered not only the uptake of other D-amino acids but also the transport of L-amino acids. The preloading with L-serine or L-leucine inhibited the uptake of D-serine and D-tyrosine into the cells as well. Selection of gap mutants for resistance to 60 120 0 D-amino acids on Pm medium. On solid media TIM E OF PRELOADING m;in containing 10 mM D-amino acids, yeast cells do FIG. 6. Time course of D-tyrosine uptake after not recover plenotypically from D-amino acid cells with unlabeled D-methionine. inhibition. Consequently, D-amino acid-resist- preload ing thewere incubated in Pm medium at 30 C XT3003A cells ant mutants could be isolated. or with (-0) addition of 5 mM unlabeled without (O) Cells of strains XT3003A and J36U mutagen- D-methionine. In indicated times, the 10-ml samples ized with ethylmethanesulfonate (19) were used were withdrawn. D-Methionine was washed out by to inoculate Pm plates supplemented with 10 centrifugation, the cells were resuspended in fresh Pm mM D-histidine, D-tyrosine, D-methionine, or medium, and the initial rate of uptake of 0.05 mM D-serine plus appropriate requirements. Single D- [ " C ]tyrosine was measured by standard procedure.

x

'5

U)

0

3

D

1

2

3

4

5

TIME ,min FIG. 5. Uptake of D-histidine and D-serine into cells preloaded with cold D-histidine. The experimental conditions were the same as described in Table 5. Symbols: 0, uptake of 0.01 mM D-histidine; A, uptake of 0.01 mM D-serine by the cells preincubated without additions (controls); 0, uptake of 0.01 mM D-histidine; and A, uptake of 0.01 mM D-serine by cells preincubated with 5 mM unlabeled D-histidine.

colonies grown after 2 to 4 days of incubation at 30 C were purified by restreaking for single-cell colonies on YPD plates. The isolated clones were replica plated from YPD master plates on five selective Pm plates containing one each of the D-amino acids and five Am plates identically supplemented. It appeared that the mutants screened as resistant to one of the D-amino acid on Pm plates could grow in the presence of any other D-amino acid on both ammonium and proline medium. In total, 15 D-amino acid-resistant mutants on both Pm and Am medium were isolated and studied in more detail. Mutants JR101 through 107 were selected in the strain XT3003A and JR111 through 116 were derived by the same procedure from the strain J36U. Two further mutants, JR108 and JR109, were isolated as spontaneous D-amino acid-resistant mutants of strain XT3003A on Pm plates containing D-histidine.

The resistance of all of thetm to D-histidine, D-serine, D-methionine, D-phenylalanine, Dtyrosine, D-tryptophan , D-leuc in e, and D-argi nine was examined on Am and Pm plates. No inhibition zone was observed on either plates. The gap mutant 2512c obtained from M. Gren-

VOL . 121, 1975

SELECTION OF AMINO AC'ID PERMEASE MUTANTS

TABLE 5. Influence of preloading the cells with D-histidine on the initial uptake rates of various amino acida Amino acid tested (0.01 mM)b&

Control

Loaded

D-Histidine D-Serine L-Histidine L-Leucine L-Citrullined

2.60 1.18 7.80 5.33 14.80

0.13

95

0.06 3.90 0.53 1.77

95 50 90

Initial uptake ratec

Ihbto Inhibition

TABLE 7. Effect of the D-amino acid resistant mutation on the initial uptake rates of L-citrulline, D-histidine, and D-serinea Initial uptake rates L-Citrulline D-Histidine D-Serine

Strain

XT3003A JR101 JR102 JR103 JR11O JR111

88

Strain XT3003A grown in Pm medium for four generations was preincubated in Pm medium for 3 h with or without 5 mM D-histidine. The cells were centrifuged, washed three times with Pm medium, and resuspended in fresh Pm medium. The uptake was measured by standard procedure. Specific radioactivities: D-histidine, 12,000 counts/min per nmol; D-serine, 6,600 counts/min per nmol; L-histidine, 18,000 counts/min per nmol; L-leucine, 14,600 counts/min per nmol; L-citruline, 11,200 counts/min per nmol. cValues expressed as nanomoles per minute per milligram (dry weight) of cells. d The concentration of L-citrulline was 0.1 mM.

567

a

(0.1 mM)

(0.1 mM)

(0.1 mM)

14.20 0.70 0.05 1.28 0.42 0.27

15.10 0.77 0.10

6.8 0.13

1.31 0.50 0.23

0.67 0.23 0.15

a Values expressed as nanomoles per minute per milligram (dry weight) of cells. Specific radioactivities as in Table 5.

sensitive strains XT3003A and J36U, it was found that D-amino acid resistance was recessive to the wild-type allele in all 15 mutants. None of the mutants complemented any other (Table 8). This result points to the conclusion that all mutations analyzed had occurred in a single cistron. A diploid constructed from the JR101 mutant TABLE 6. Inhibition of D-serine and D-tyrosine uptake and 2512c gap mutant was resistant to D-amino after preloading with L-amino acidsa acids on Pm and Am plates. The tetrad analysis of eight asci was carried out. All haploid segreInitial upInitial upAmino acid take rate of Inhibitake rate of Inhibigants of this cross were D-amino acid resistant used for ford used tion (%) D-tyrosine tion (% D-serine on either Am or Pm medium. These results preloading (0.01 mm)" (0.01 MM)b indicated that mutants selected as D-amino acid resistant on Pm plates are allelic with the None 1.14 0.79 L-Serine 0.19 gap mutants. Therefore, slection for D-amino 83 0.18 77 L-Leucine 0.18 84 0.14 82 acid resistance on minimal proline medium is a positive selection for general amino acid pera Experimental conditions as in Table 4. b Values expressed as nanomoles per minute per mease mutants. Regulatory mutants selected for resistance milligram (dry weight) of cells. Specific radioactivities

as

in Table 2.

son was also resistant to all of these D-amino acids, both on Am and Pm medium. As it is shown in Table 7, the mutants showed a reduced rate of D-histidine and D-serine uptake. The mutants had only 1 to 10% of the wild-type activity. Also, the uptake of L-citrulline was reduced to the same level as that of D-amino acid uptake. Since L-citrulline is transported by the general amino acid permease and the uptake of this amino acid is significantly decreased in gap mutants (8), the reduced uptake of L-citrulline in D-amnino acid-resistant mutants suggested that D-amino acids are transported by the general amino acid permease. Complementation tests were used to analyze these mutants. In crosses with D-amino acid-

TABLE 8. Complementation tests of D-amino acidresistant mutants on Pm mediuma a uraa ad2.1

J

J36U JR111 JR112 JR113 JR114 JR115 JR

XT3003A

JR101

-

_

_

_

_

_

+ + +

+ + +

+ + +

+

+ + + +

+ + + + +

+ + + + +

JR104

-

+ + + +

JR105

-

JR106

+

-

-

+ + +

+ + + + +

-

+

+

JR102 JR103

JR107 JR108

JR109

-

-

-

+ + + + + +

+ + + +

+ + + + +

The complementation test was carried out on Pm plates supplemented with 10 mM D-histidine. Symbols: +, growth in the presence of D-histidine; -, lack of growth. All diploids grew on Pm plates. a

J. BACTERIOL.

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568

to D-amino acids on Am medium. The mutants isolated as D-amino acid resistant on Am medium could be divided into two classes. Strains of one class were the gap mutants resistant to D-amino acids on either Am or Pm medium. Mutants of the second class were resistant to D-amino acids on Am medium but remained sensitive on Pm medium. They behaved in the same way as the strain 21278b. The mutant JR211 was obtained by ethylmethanesulfonate mutagenesis of strain J36U and selection for the D-amino acid resistance on Am plates containing uracil and 10 mM D-histidine. Mutant JR211, similar to strain 2:1278b, showed the conditional resistance to D-amino acids, i.e., it was resistant only when grown in the presence of ammonium ions. As shown in Table 4, strains M1278b and JR211 grown on ammonium sulfate as nitrogen source had lower initial rates of D-serine uptake than did cells grown on L-proline. The genetic analysis of strains JR211 and 1278b was carried out to find if these two mutants are allelic. The two strains were crossed to D-amino acid-sensitive strains XT3003A and J36U, respectively. As shown in Table 9, in both cases the diploid strains were resistant to D-amino acids on Am medium and sensitive on Pm medium. This meant that resistance to D-amino acids in the presence of ammonium ions was dominant over the sensi-

tivity. Tetrad analysis of some of the crosses presented in Table 9 was carried out. The spore colonies were tested for mating type, nutritional requirements, and their ability to grow on Am and Pm media supplemented with 10 mM D-histidine. In the crosses 2M1278b x J36U and JR211 x XT3003A, the alleles producing the phenotype of D-amino acid resistance on Am medium showed 2:2 segregation. All spores resulting from dissection of 10 asci of the cross 2:1278b x TABLE 9. Dominance of the amc+ genotype in diploidsa Haploidsa a

J36U

JR211

JR212

JR111

2512c

JR211 were resistant to D-amino acids on Am and sensitive on Pm medium. Mating type and nutritional requirements in all tetrads showed 2:2 segregation. The above result indicated that strains Z1278b and JR211 were allelic for Damino acid resistance on Am medium. In the previous section it was shown that D-amino acid resistance on both Am and Pm medium was caused by mutation in general amino acid permease locus gap. To find out if the mutation affecting the activity of general amino acid permease only in the presence of ammonium ions is in the gap locus too, the tetrad analysis of the diploid resulting from a cross between strains JR101 and JR211 was done. Eight tetrads were examined and the analyses of these tetrads (see Table 11) show that these two mutations segregate independently. The gene designation amc+ is given to strains like Z1278b and JR211 which are resistant to D-amino acids on Am but not on Pm plates. Correspondingly, strains 'that are sensitive to D-amino acids on Am plates, e.g., XT3003A, carry amc- alleles. The double mutant amc+ gap was constructed from the cross JR101 x JR211 (ade2-1 amc- gap x a ura amc+. Growth tests of haploids (Table 10) did not permit differentiation between gap amc+ and gap amc- mutations. As shown in Table 9, the analysis of growth of diploids, resulting from crosses with the amc+ and amc- on both Am plus D-histidine and Pm plus D-histidine, permitted the distinction between these two genotypes. As is clear from data presented in Table 9, the mutant 2512c (derived from Z1278b [8]) in the cross with strains XT3003A and JR101 behaved like mutant JR212 amc+ gap. Results of tetrad analysis of the above crosses are shown in Table 11. The presence of all three classes of tetrads, parental ditype (PD), nonparental ditype (NPD), and tetratype (TT) in the ratio close to PD:NPD:TT 1:1:4, indicates that two mutations amc and gap are affecting different and nonlinked genetic loci. TABLE 10. Distinctive tests for wild type and mutants

Am Pm Am Pm Am Pm Am Pm Am Pm

Growth of mutants Medium (supplemented with

XT3003A

-

-

Z1278b _ JR101

-

-

+ + ±

-

+ +

-

±

-

-

-

-

+

-

-

+ +

-

+

-

+

±

+

+

The growth test was carried out on Am and Pm plates supplemented with 10 mM D-histidine. Symbols as for Table 8. The diploids were obtained by isolating zygotes with a micromanipulator. a

auxotrophic requiremenits)

amc-

Am Am + 10 mM D-histidine Pm Pm + 10 mM D-histidine

amc+

amc-

amc+

gap

gap

_+

+

-

+ + +

+

+

+ +

-

-

+

+

+

VOL. 121, 1975

SELECTION OF AMINO ACID PERMEASE MUTANTS

TABLE 11. Tetrad data indicating independent segregation of gap and amc loci gap and amc

Diploid

segregationa

Genotype PD

XT3003A

af ade2, amc-

I

JR212 XT3003A

a ura amc+ gap a ade2, amc-

2512c JR101

a gap amc+ a ade2 lgap amc-

JR211

a ura amc+

NPD

1 ~~~~~~2

Tl 4

1

1

5

2

0

6

a Segregation for auxotrophic requirements and mating type was 2:2. PD, Parental ditype; NDP,

nonparental ditype; TT, tetra type. DISCUSSION The data presented in this paper show that cells of the yeast S. cerevisiae are sensitive to D-stereoisomers of natural amino acids. Individual D-amino acids differ only slightly in their ability to inhibit the growth of yeast. Of all D-amino acids tested, D-histidine appeared to be the most effective inhibitor. Studies on D-amino acid uptake indicate that they enter the cell by a single transport system. The differences in the degree of inhibition uptake of D-amino acids by D-serine and D-tyrosine and of the differences in Km values determined for the uptake of D-histidine and D-serine possibly resulted from differences in the affinity of particular D-amino acids for the transport system. The notion that all D-amino acids tested are transported by the same transport system was further substantiated by the fact that mutants isolated as resistant to one Damino acid were resistant to all of them. The decreased uptake of D-amino acids observed in the presence of L-amino acids suggested that D-amino acids are transported into yeast cells by a system common for both stereoisomers. Data presented by Grenson et al. (8) suggested that D-methionine and D-alanine might be transported by the general amino acid permease. The fact that L-citrulline, the amino acid transported essentially only by the general amino acid perinease (8), strongly inhibited the uptake of D-serine was an indication that this permease can be a transport system for D-amino acids. In fact, the genetic analysis of mutants resistant to D-amino acids on L-proline as a nitrogen source proved that they are allelic to the gap mutant of Grenson et al. (8).

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The experiments presented demonstrate that a D-amino acid once concentrated within the cells inhibits a further uptake of gap substrates. The cells preloaded with D-histidine showed decreased uptake not only of D-histidine but also of D-serine; D-methionine preloaded cells showed a decrease of D-tyrosine uptake. The preloading with D-amino acids of yeast cells diminished the uptake also of L-amino acids. The reverse situation was observed as well. Previous exposure to L-amino acid reduced D-amino acid uptake. This kind of regulation of the activity of amino acid transport systems by the amino acid accumulated inside the cell has been described for numerous amino acid permeases (1, 3, 12, 14, 15, 16) and is known as transinhibition. It was reported by Crabeel and Grenson (3) that in S. cerevisiae the uptake of L-histidine is regulated by L-histidine accumulated within the cells. Data presented in this paper suggest that in yeast the uptake of other amino acids, not only of L-histiditie, is regulated by transinhibition. Since the D-amino acids and most of L-amino acids are substrates of the general amino acid permease, one can conclude that its activity is regulated by transinhibition. Grenson et al. (8) reported that the activity of the general amino acid permease in S. cerevisiae is inhibited by ammonium ions. A comparison of two strains, S288C and 21278b, showed that they differed in their sensitivity to D-amino acids depending on nitrogen source in growth medium. Both strains were sensitive to D-amino acids (to the same extent) when grown on L-proline as a nitrogen source, whereas in medium containing ammonium ions only strain S288C was sensitive. The resistance of strain 21278b to D-amino acids on Am medium was caused by almost complete elimination of Damino acid uptake. Since the D-amino acids are transported by the general amino acid permease system, the observed difference between the two strains S288C and 21278b is a result of different sensitivity of the same permease to ammonium ions. From the D-amino acid-sensitive strain J36U, a mutant JR211 was obtained which behaved identically to 21278b in the sense that it was sensitive to the D-amino acids on Pm but not on Am medium. JR211 had reduced uptake of D-amino acids in the presence of ammonium ions (Table 4). The inhibitory effect of ammonium ions on a proline permease was observed in S. chevalieri (18) and S. cerevisiae (8). It was suggested that, in Aspergillus nidulans, formation of an acidic amino acid permease is regulated by a mechanism involving repression by ammonia or prod-

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ucts of its metabolism (17). In Penicillum chrysogenum, the general amino acid permease is inhibited by ammonium ions. According to the authors, the intracellular NH,+ acts as a feedback inhibitor of this permease (1). Although the target of ammonium action on the amino acid permeases in fungi has not yet been identified, one can assume that ammonium ions or a product of their metabolism can regulate the uptake of amino acids by cells. The finding that S. cerevisiae cells are sensitive to D-amino acids and that the toxicity of D-amino acids depends on their uptake permits differentiation between strains with NH,+-sensitive or NH4+-insensitive general amino acid permease by checking the sensitivity of the strains to D-amino acids on Am and Pm medium. The genetic analysis of strains in which the activity of general amino acid permease is affected by ammonium ions showed that the amc gene responsible for this phenotype is not linked with the gap gene. The mutants resistant to D-amino acids on Am and Pm medium carry a gap mutation. Since gap mutants were recessive and not one of the isolated mutants complemented any other, it can be concluded that the lesions are in the same cistron controlling a polypeptide chain, the function of which is essential for the permease in S. cerevisiae. Isolation of mutants resistant to D-amino acids on both Am and Pm medium is a positive selection for general amino acid permease mutants. The fact that sensitivity of the general amino acid permease to ammonium ions is dependent on the presence of the amc+ gene suggests that the amc product in combination with ammonium ions is responsible for production of some ammonium metabolite, which affects the general amino acid permease. ACKNOWLEDGMENT This work was supported by the Polish Academy of Sciences within the project 09.3.1.

LITERATURE CITED 1. Benko, P. V., T. C. Wood, and

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H. Segel. 1969.

Multiplicity and regulation of amino acid transport in Penicillium chrysogenum. Arch. Biochem. Biophys. 129:498-508.

2. Bussey, H., and H. E. Umbarger. 1970. Biosynthesis of branched-chain amino acids in yeast: a leucine-binding component and regulation of leucine uptake. J. Bacteriol. 103:277-285. 3. Crabeel, M., and M. Grenson. 1970. Regulation of histidine uptake by specific feedback inhibition of two histidine permeases in Saccharomyces cerevisiae. Eur. J. Biochem. 14:197-204.

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4. Gits, J. J., and M. Grenson. 1967. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. III. Evidence for a specific methionine-transporting system. Biochim. Biophys. Acta 135:507-516. 5. Grenson, M. 1966. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. II. Evidence for a specific lysine-transporting system. Biochim. Biophys. Acta 127:339-346. 6. Grenson, M., M. Crabeel, J. M. Wiame, and J. Bechet. 1969. Inhibition of protein synthesis and stimulation of permease turnover in yeast. Biochem. Biophys. Res. Commun. 30:413-419. 7. Grenson, M., and C. Hennaut. 1971. Mutation affecting activity of several distinct amino acid transport systems in Saccharomyces cerevisiae. J. Bacteriol. 105:477-482. 8. Grenson, M., C. Hou, and M. Crabeel. 1970. Multiplicity of the amino acid permease in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease. J. Bacteriol. 103:770-777. 9. Grenson, M., M. Mousset, J. M. Wiame, and J. Bechet. 1966. Multiplicity of the amino acid permease in Saccharomyces cerevisiae. I. Evidence for a specific arginine transporting system. Biochim. Biophys. Acta

127:325-338. 10. Halvorson, H. O., and G. N. Cohen. 1958. Incorporation des amino acides endogenes et exogenes dans les protbines de la levure. Ann. Inst. Pasteur 95:73-87. 11. Hawthorne, D. C., and R. K. Mortimer. 1960. Chromosome mapping in Saccharomyces: centromerlinked genes. Genetics 45:1085-1110. 12. Hunter, D. R., and I. H. Segel. 1973. Control of the general amino acid permease of Penicillium chrysogenum by transinhibition and turnover. Arch. Biochem. Biophys. 154:387-399. 13. Lacroute, F., A. Pierard, M. Grenson, and J. M. Wiame. 1965. The Biosynthesis of carbonoyl phosphate in Saccharomyces cerevisiae. J. Gen. Microbiol. 40:127-142. 14. Pall, M. L. 1971. Amino acid transport in Neurospora crassa. IV. Properties and regulation of a methionine transport system. Biochim. Biophys. Acta 233:201-214. 15. Pall, M. L., and K. A. Kelly. 1971. Specificity of transinhibition of amino acid transport in Neurospora. Biochem. Biophys. Res. Commun. 42:940-947. 16. Ring, K., W. Gross, and E. Heinz. Negative feedback regulation of amino acid transport in Streptomyces hydrogenans. Arch. Biochem. Biophys. 137:243-252. 17. Robinson, J. H., C. Anthony, and W. T. Drabble. 1971. Activity and regulation of an acidic amino acid permease in Aspergillus nidulans. Biochem. J. 124:75P. 18. Schwencke, J., and N. Magana-Schwencke. 1969. Derepression of a proline transport system in Saccharomyces chevalieri by nitrogen starvation. Biochem. Biophys. Acta 173:302-312. 19. Sherman, F., G. R. Fink, and C. W. Lawrence. 1971. Methods in yeast genetics. Laboratory manual. Cold Spring Harbor, New York. 20. Sorsoli, W. A., K. D. Spence, and L. W. Parks. 1964. Amino acid accumulation in methionine-resistant Sac-

charomyces cerevisiae. J. Bacteriol. 88:20-24. 21. Surdin, Y., W. Sly, J. Sire, A. M. Bordes, and H. De Robinchon-Szulmajster. 1965. Propertietes et controle genetique du systeme d'accumulation des acides

amines chez Saccharomyces cerevisiae. Biochim. Biophys. Acta 107:546-566. 22. Zenk, M. H., and J. M. Schmitt. 1965. Reinigung und Eigenschaften von Acetyl-CoA Daminosaure a-NAcetyltransferase aus Hefe. Biochem. Z. 342:54-65.

Positive selection of general amino acid permease mutants in Saccharomyces cerevisiae.

It was found that D-stereoisomers of natural amino acids inhibit the growth of Saccharomyces cerevisiae cells. Kinetic and genetic evidence showed tha...
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