Archives of
Microbiology
Arch. Microbiol. 107, 207-214 (1976)
9 by Springer-Verlag 1976
Free Tryptophan Pool and Tryptophan Biosynthetic Enzymes in Saccharomyces cerevisiae PETER A. FANTES*, LILIAN M. ROBERTS, and RALF HUETTER Mikrobiologisches Institut der Eidgen6ssischen Technischen Hochschule, Universit/itsstr. 2, CH-8006 Ziirich, Switzerland
Abstract. The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties. The tryptophan pool of wild type cells growing in minimal medium is 0.07 gmole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth" medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool. Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by L-tryptophan or by the analogue DL-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced ~ryptophan pool, but no derepression of indoleglycerolphosphate synthase was found. A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.
* Present address: Department of Zoology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JN, Scotland. Abbreviations. CDRP = 1-(o-carboxyphenylamino)-l-deoxyribulosephosphate; paba = paraaminobenzoic acid; PRA = N(5'-phosphoribosyl)-anthranilate; tRNA = transfer ribonucleic acid; trpl to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes.
Key words." Anthranilate synthase - Cell permeabilisation - Indoleglycerolphosphate synthase - Saccharomyces cerevisiae - Tryptophan biosynthetic enzymes - Tryptophan pool.
A substantial body of information now exists about the regulation of biosynthetic pathways in yeast and other fungi (De Robichon-Szulmajster and SurdinKerjan, 1971; Gross, 1969). Much of the published work describes the variation in enzyme levels under different nutritional conditions, and the effect of regulatory mutations on them. In only a few cases, however, have the internal concentrations of intermediates and the end product of the pathway been measured (Masselot and De Robichon-Szulmajster, 1974; Ramos et al., 1970; Rasse-Messenguy and Fink, 1973). This report is concerned with the interactions between the tryptophan pool and tryptophan biosynthetic pathway, using different conditions of growth and mutants with altered regulatory properties. A simplified scheme of tryptophan biosynthesis is given in Figure 1. The great majority of tryptophan is probably used for protein synthesis, as (i) the amount needed for nicotinic acid biosynthesis is very small, and this vitamin is present in the medium, (ii) exogenous tryptophan is used only slowly as nitrogen source, indicating a low rate of tryptophan degradation in Saccharomyces cerevisiae (Shetty and Gaertner, 1973). The enzymes assayed in this work, anthranilate synthase and indoleglycerolphosphate synthase, first and fourth enzymes respectively of the tryptophan pathway, are known to occur as an aggregate in vitro, and are thought to do so in whole cells (Htitter and DeMoss, 1967). As their regulation appears to be coordinate (Schiirch et al., 1974), we are able to assay the aggregate in mutants with altered anthranilate synthase by measuring indoleglycerolphosphate synthase.
208
Arch. Microbiol., Vol. 107, No. 2 (1976) REPRESSION
f
INHIBITION
Chorismate 9
~. Anthranilate
~ PRA
~ CDRP :
phe, tyr
paba
anthranilate synthase~ trp 2, tr_~p_3
. ~ Indoleglycerol : phosphate i
anthranilate phosphori- indoleglycerolphosphorihosylbosyl phosphate transferase anothg2n~I2te synthase " ~ 4
~
p
3
tryptophan synthase
Tryptophan I I I
NAD
~ Tryptophanyl-tRNA
1
Protein
trp 5
enzyme aggregate Fig. 1. Tryptophan biosynthesis in Saccharomyees cerevisiae
F r o m p r e v i o u s studies it is k n o w n t h a t starving a t r y p t o p h a n a u x o t r o p h for t r y p t o p h a n leads to derep r e s s i o n ( M i o z z a r i , J., u n p u b l i s h e d d a t a f r o m this labor a t o r y ) a n d t h a t the t r y p t o p h a n a n a l o g u e DL-5-methylt r y p t o p h a n causes a r e d u c t i o n in g r o w t h r a t e a n d a d e r e p r e s s i o n o f the t w o e n z y m e s a n t h r a n i l a t e synthase a n d i n d o l e g l y c e r o l p h o s p h a t e synthase b y a f a c t o r o f a b o u t 2.5 (Schtirch et al., 1974). T o gain a b e t t e r insight into the r e g u l a t o r y process, the t r y p t o p h a n p o o l a n d e n z y m e activities have been m e a s u r e d here in e x p o nentially g r o w i n g cells, a n d c o n d i t i o n s w h e r e g r o w t h is u n b a l a n c e d , such as s t a r v a t i o n o f a t r y p t o p h a n a u x o t r o p h for t r y p t o p h a n , have been a v o i d e d . R e d u c tion in the t r y p t o p h a n a v a i l a b l e to the Cells was b r o u g h t a b o u t b y using m u t a n t s with low e n z y m e activity, a l o n g the lines o f the " m o d u l a t i o n " experim e n t s o f K a c s e r a n d Burns (1968). I n a d d i t i o n to the wild t y p e strain, two classes o f r e g u l a t o r y m u t a n t s have been s t u d i e d in o u r laboratory: those showing altered feedback parameters o f a n t h r a n i l a t e s y n t h a s e (Schtirch et al., 1974), a n d those w h i c h h a v e lost the ability t o d e r e p r e s s (Schiirch et al., 1974) or repress the synthesis o f at least the t w o t r y p t o p h a n enzymes a s s a y e d here.
pH adjusted to 4.0 with KOH. The use of buffered medium reduces variation in the inhibition of growth rate by 5-methyltryptophan. Media and supplements except 5-methyltryptophan were sterilised by autoclaving separately at 120~ for 20 min; 5-methyltryptophan was filter sterilised. Concentrations of 5-methyltryptophan added to the growth media are indicated in the text. Cultures were grown at 30~C, liquid cultures on a rotary shaker at 150 strokes per minute. Growth was followed turbidimetrically at 546 nm with a 1 cm light path, and dry weights related to this using a calibration curve. The specific growth rate, #, is defined as = (ln x2-1n Xt)/(t2-tl). One unit of optical density (OD546 of 1.0) corresponds to approx. 1.5 9 10l~ cells per ml, which equal 1.1 g wet weight, 165 mg dry weight or 80 mg protein.
MATERIALS
Enzyme Assays 1. Anthranilate synthase activity was determined using a "stop assay" (Egan and Gibson, 1970). The reaction was started by adding 0.2 ml of permeabilised cell suspension suitably diluted in KMG buffer to the assay mix, which contained, in a final volume of 1.0 ml: 0.1 mmole potassium phosphate, pH 7.6; 5 ixmole MgSO4; 20 gmole L-glutamine; 0.1 gmole chorismic acid. The assay was run for 20 rain at 37~ and stopped by adding 0.1 ml i N HC1. The product, anthranilic acid, was extracted in 4 ml ethyl
AND METHODS
Chemicals. The tryptophan analogueDL-5-methyltryptophan (puriss. grade) was obtained from Fluka AG, Buchs SG, Switzerland. Escherichia coli tryptophanase (grade II, lot 43C-6910, activity quoted per mg: 30 pg indole released from L-tryptophan per 10 min at pH 8.3 and 37~ was purchased from Sigma Chemical Co., St. Louis, Missouri. Chorismic acid was made according to Gibson (1970), and CDRP [1-(o-carboxyphenylamino)-l-deoxyribulose phosphate) was made by the method of Smith and Yanofsky (1962). Other chemicals were of analytical grade. Media and Growth Conditions. YEPD medium (1 ~ oxoid yeast extract, 2 ~ oxoid peptone, 2 ~ glucose plus 2 ~ agar for solid medium) was used as complete medium and for maintenance of strains. The minimal medium used was MV medium (0.145 ~o Difco yeast nitrogen base without amino acids and ammonium sulphate, 0.525 G ammonium sulphate, 2 ~ glucose) with 1 G succinic acid,
Permeabilisation of Cellsfor Enzyme Assays. Cultures were harvested at an optical density of approximately 1.0 by centrifugation at 2-4~ After resuspension in "KMG" buffer (0.1 M potassium phosphate, pH 7.6, containing 5 mM magnesium sulphate and 20 mM L-glutamine as stabilisers for anthranilate synthase), the cells were again centrifuged, and resuspended to 100 mg wet weight per ml in KMG. Dimethylsulphoxide was added to a final concentration of 40~ (v/v) and after standing for 10 min at 0~ the cells were centrifuged and resuspended in KMG. The washing was repeated and the final resuspension made in KMG to 100 mg wet weight per ml (Adams, 1972). Enzyme activities were stable for at least 6 h under these conditions. Protein was estimated in the permeabilised cell preparations by the biuret method described by Herbert et al. (1971). Control experiments showed that the permeabilisation has no detectable effect on the protein content of the cells.
t Anthranilate synthase [chorismate pyrt/vate lyase (amino accepting) ; EC 4.1.3.27] refers to the activity measured with glutamine as amino donor; anthranilate phosphoribosyltransferase [N-(5'phosphoribosyl)-anthranilate pyrophosphate phosphoribosyltransferase; EC 2.4.2.18]; indoleglycerolphosphate synthase [1-(2'-carboxyphenylamino)- 1-deoxyribulose- 5-phosphate carboxylase-[cyclizing); EC 4.t.1.48]; phosphoribosyl-anthranilate isomerase [N(5'-phosphoribosyl)-anthranilate isomerase]; tryptophan synthase [L-serine hydrolyase (adding indole); EC 4.2.1.20].
P. A. Fantes et al. : Tryptophan Pool Size and Enzyme Levels in Saccharomycescerevisiae
209
Table 1. Strains of Saecharomyees cerevisiae Strain
Growth rate in MV medium
~ Growth inhibition by 5 • 10 -4 M DL-5methyltryptophan (liquid culture)
Growth sensitivity to 10 -~ M DL-5fluortryptophan (solid medium)
Description (other phenotypic characters)
X2180-1A
0.310
29
Sensitive
Wild type strain
RH 424
0.272
50
Sensitive
Anthranilate synthase reduced and feedback-supersensitive (maps in trp2) (Sch/ireh et al., 1974)
RH 428 RH 482
0.268 0.276
47 52
Sensitive Sensitive
RH 487 RH 595
0,265 0.291 "
65 0
Sensitive Resistant
Non-derepressible for tryptophan enzymes (Schtirch et al., 1974) Anthranilate synthase greatly reduced and feedback-supersensitive (maps in trp2) (Schfirch et al., 1974) Non-derepressible for tryptophan enzymes (Schiirch et al., 1974) Slightly feedback-resistant anthranilate synthase; derepressed enzyme levels
RH 596
0.257
20
Sensitive
RH 597 RH 598
0.281 0.233
0 0
Resistant Resistant
acetate, and estimated by its fluorescence measured at 340 nm excitation, 408 nm emission, on an Aminco-Bowman Spectrofiuorimeter (Egan and Gibson, 1970). Indoleglycerolphosphate synthase was assayed as described previously (Schtirch et al., 1974), using permeabilised cells instead of cell-free extract.
Extraction of Soluble Pool of Cells. For most experiments, four parallel cultures of 400 ml MV medium plus supplements were used for each experimental condition. The cultures were harvested by vacuum filtration on Whatman GF/C fiiters supported in Millipore 47 mm filtration units, and washed twice with 25 ml ice-cold water. Filters were removed and all four immersed in 10 ml icecold (60 ~ (v/v) ethanol. After 15 rain, a portion of the extracted suspension was filtered through a GF/C filter and washed with 10 ml 60~o ethanol. The filtrate was stored at - 2 5 ~ Extracts were evaporated to dryness on a rotary evaporator at 35-40~ and the material taken up in a small volume (1 - 5 ml) of water.
Derepressed enzyme levels; derived from RH 595 by recombination Feedback-resistant anthranilate synthase (maps in trp2) Feedback-resistant anthranilate synthase (maps in trp2)
(up to 50-fold) of 5-methyltryptophan. Three different sample volumes were assayed for each determination. Media were obtained from Difco, and the organism used was Lactobacillus plantarum, ATCC 8014.
Strains of Saccharomyces cerevisiae. All the strains used were derived by mutation and recombination from the isogenic wild type strains X2180-1A (a mating type) and X2180-1B (c~ mating type). The main characteristics of the strains are presented in Table 1. Strain RH 595 was isolated after N-methyl-N'-nitro-N-nitrosoguanidine treatment of the wild type strain X2180-1A as a 5-fluortryptophan resistant mutant. Strain RH 597 was isolated as a spontaneous 5-fluor-tryptophan resistant mutant from the same wild type strain. Strain RH 598 was isolated as a feedback resistant segregant from strain RH 446, a 5-methyltryptophan resistant revertant of strain RH 487.
Estimation of Tryptophan. Two methods were used, which gave
RESULTS
results within 20 ~ of one another. (i) Enzymatic assay (DeMoss, 1962). This method was the more accurate of the two, but less sensitive. It couId not be used to determine tryptophan in the presence of 5-methyltryptophan, as the latter interferes. A sample of the material to be assayed was incubated with tryptophanase in a buffer system with the following composition in a final volume of 1.2 ml: 0.144 mmole potassium phosphate, pH 7.8; 0.24 gmole dithiothreitol; 0.072 gmole pyridoxal phosphate; 0.2 mg tryptophanase preparation. The test sample was overlaid with 1.0 ml toluene. Assay tubes were stoppered and incubated in a shaking water bath at 37~ for 2h. Then 0.2ml 1 N NaOH was added to each tube, and 0.5 ml of the toluene layer assayed for the reaction product, indole, by the :method of Smith and Yanofsky (1962). Indole and tryptophan standards were included in each set of assays. Recovery of known amounts of L-tryptophan was between 90 and 100 ~. (ii) Microbiological assay. The procedure followed is based on that described in the Difco Manual (1953). The main difference is that growth was measured turbidimetrically between 12 and 15 h after inoculation, rather than acidimetrically. This procedure made it possible to assay tryptophan in the presence of a large excess
Tryptophan Pool in the Wild Type Strain A s s h o w n in T a b l e s 2 - 5 , t h e t r y p t o p h a n p o o l in w i l d t y p e cells g r o w i n g in m i n i m a l m e d i u m is n e a r 0.07 g m o l e p e r g d r y w e i g h t . T h e a d d i t i o n o f t r y p t o p h a n o r m e t a b o l i c p r e c u r s o r s to t h e m e d i u m b y p a s s e s the control by feedback inhibition of anthranilate s y n t h a s e , a n d t h e t r y p t o p h a n p o o l is g r e a t l y i n c r e a s e d u n d e r t h e s e c o n d i t i o n s ( T a b l e 2). T h e p r e s e n c e o f s u c h a h i g h t r y p t o p h a n p o o l d i d n o t , h o w e v e r , l e a d to repression of the tryptophan enzymes.
Tryptophan Pool and Enzyme Activities in Mutants with Altered Feedback Properties of Anthranilate Synthase T w o classes o f m u t a n t s w i t h a l t e r e d f e e d b a c k p r o p e r ties in a n t h r a n i l a t e s y n t h a s e w e r e a n a l y s e d , f e e d b a c k
210
Arch. Microbiol., Vot. 107, No. 2 (1976)
Table 2. Effectof growth medium on tryptophan pools and enzyme activities in wild type strain X2180-1A Growth medium
Growth rate
MV MV + Tryptophan ~ MV + Indol& MV + Anthranilic acid"
0.310 0.310 0.307 0.279
Specific enzyme activities (nmole mg protein- 1 min -1) Anthranilate synthase
Indoleglycerolphosphate synthase
1.40 1.10 1.38 1.20
2.15 2.33 2.60 1.85
Tryptophan pool (lamole g dry weight-i)
0.062 0.708 3.40 1.80
Supplements added at 20 lag m1-1. Table 3. Tryptophan pools and enzyme activities in mutants with altered feedback sensitivity Strain
X2180-1A RH 597 RH 598 RH 424 RH 482
Growth rate in MV medium
0.309 0.281 0.233 0.272 0.276
Specific enzyme activities (nmole mg protein-t min- i) Anthranilate synthase
Indoleglycerolphosphate synthase
1.32 1.42 1.38 0.33 0.05
2.23 2.77 1.83 1.62 2.62
Feedback sensitivity"
Tryptophan pool (pmole g dry weight-1)
10-5 > 10.3 > 10-3 2x 10 6 3 x 10-6
0.065 3.75 3.12 0.036 0.037
Tryptophan pools less than 0.1 lamole per g dry weight were determined a minimum of two times. Values given are means. a Concentration of L-tryptophan which inhibits the activity by 50 ~ under the conditions described in "Materials and Methods". Table 4. Tryptophan pool and enzyme activities in regulatory mutants Strain
X2180-1A RH 595 RH 596 RH 487 RH 428
Growth rate in MV medium
0.281 0.291 0.257 0.265 0.268
Specific enzyme activities (nmole mg protein-1 min 1) Anthranilate synthase
Indoleglycerolphosphate synthase
1.32 3.03 2.65 1.07 1.00
2.25 4.42 3.63 1.38 1.20
Tryptophan pool (pmole g dry weight -t)
0.072 2.70 1.22 0.095 0.069
Tryptophan pools less than 0.1 pmole per g dry weight were determined a minium of two times. Values given are means.
resistant and feedback supersensitive strains. It was possible that although externally supplied t r y p t o p h a n or its metabolic precursors w o u l d n o t repress the biosynthetic p a t h w a y , internally synthesised t r y p t o p h a n might do so: differential effects o f externally supplied and endogenously synthesised t r y p t o p h a n have been r e p o r t e d in Neurospora crassa (Matchett et al., 1968), and yeasts m a y contain a m i n o acids in several physiologically distinct pools (Bearden and Moses, 1972; Cowie and McClure, 1959). To test this possibility, we m a d e use o f m u t a n t s with altered anthranilate synthase which is less sensitive to inhibition by tryptop h a n than the wild type strain. Table 3 shows that
the t r y p t o p h a n p o o l in these m u t a n t s is very high, higher than that obtained in the wild type strain under m o s t conditions. The activity o f indoleglycerolphosphate synthase is however no lower than in the wild type strain. These experiments show that if t r y p t o p h a n itself is the molecule corresponding to the corepressor in bacterial systems (Epstein and Beckwith, 1968), then the t r y p t o p h a n biosynthetic p a t h w a y in Saccharomyces cerevisiae is fully repressed during growth on minimal medium. I f t r y p t o p h a n pool size is a controlling factor in the regulation o f its o w n biosynthesis at the level o f
P. A. Fantes et al. : Tryptophan Pool Size and Enzyme Levels in Saccharomyces cerevisiae
211
Table 5. Effect of 5-methyltryptophan on tryptophan pools and enzyme levels in wild type strain X2180-1A and the mutant strains RH 428 and RH 487 Growth medium
Growth rate
Specific enzyme activity (nmole mg protein- ~ min-1) Anthranilate synthase
Indoleglycerolphosphate synthase
Tryptophan pool (gmole g dry weight -1) (mean values and standard error)
Wild type strain : MV MV + DL-5-methyltryptophan (5 x 10 -4 M)
0.295 0.194
1.40 3.22
2.15 6.50
0.069 • 0.004 0.073 _+ 0.011
Mutant strain RH 428 : MV MV + DC-5-methyltryptophan (10 .4 M)
0.296 0.067
1.48 0.85
1.70 1.12
0.068 ___ 0.002 0.039
Mutant strain RH 487: MV MV + DL-5-methyltryptophan (10 .4 M)
0.264 0.073
1.16 1.36
1.50 1.76
0.11 0.05
Where standard errors are given the values for the tryptophan pool are based on ten determinations (both enzymatic and microbiological) for MV medium, and four determinations (microbiological only) for MV + 5 MT medium.
enzyme synthesis, then a reduction in pool size should lead to derepression of the pathway. To test this, we used mutants with decreased anthranilate synthase activity which was also more sensitive to inhibition by tryptophan than the enzyme of the wild type strain. Table 3 shows that the pool in these mutants is reduced by about one half, which is a smaller decrease than the 75 ~ and 96 ~ reduction in anthranilate synthase activity in strains RH 424 and R H 482 respectively. Despite the smaller tryptophan pools in these mutants, there is no evidence for derepression of indoleglycerolphosphate synthase in either. It should be pointed out that both strains have exponential growth rates essentially the same as the wild type strain, in spite of their low biosynthetic capacity.
Tryptophan Pool and Enzyme Activity in Mutants with Altered Regulation of Tryptophan Biosynthetic Enzymes Two types of mutants of this class have been isolated. One type of regulatory mutant has only recently been isolated, which maintains higher enzyme levels than the wild type strain under all growth conditions tested and may therefore be considered "derepressed". The main characteristics are summarised in Table i. Pools and enzyme levels were determined for strains R H 595 and R H 596, and the results in Table 4 show an increase in the two enzyme activities and the pool size. This shows that the high tryptophan pool is a result of the increased enzyme levels, rather than the enzyme levels being high as a result of tryptophan starvation. Strain R H 595 has a higher pool than strain R H 596 pre-
sumably as a consequence of the relative insensitivity of its anthranilate synthase to tryptophan inhibition. Mutants of the other type have been described previously (Schtirch et al., 1974). They have lost the ability to derepress the biosynthetic enzymes in response to either the presence of 5-methyltryptophan or deprivation of tryptophan when combined with tryptophan auxotrophy in a double mutant. The results in Table 4 show that the inability to derepress is not due to a permanently high internal tryptophan pool.
Effect of 5-Methyltryptophan on Tryptophan Pool and Enzyme Activities As described in a previous report (Schfirch et al., 1974), inclusion of DL-5-methyltryptophan in the growth medium produces an inhibition of growth of the wild type strain which under the conditions described here is about 30 ~, and leads to a derepression of the biosynthetic enzymes of about 2.5-fold (Table 5). The analogue inhibits anthranilate synthase in extracts and permeabilised cells to about the same degree as tryptophan, and these results led Schfirch et al. (1974) to the following hypothesis: 5-methyltryptophan inhibits anthranilate synthase and causes a reduction in tryptophan biosynthesis, which leads to a reduced tryptophan pool. The reduction in pool size then effects a derepression of the biosynthetic pathway. To test this hypothesis more directly, the tryptophan pool in cells growing in the presence of the analogue was determined. The results (Table 5) show that there is little, if any difference between pool size of wild type cells grown in the presence or absence of this
212 analogue. Any reduction in the tryptophan pool caused by the analogue is smaller than that produced by a defective anthranilate synthase (see Table 3). For mutants lacking the ability to derepress the tryptophan biosynthetic enzymes in response to 5-methyltryptophan, strains RH 428 and RH 487, the situation is different. In this case 5-methyltryptophan causes a significant reduction in tryptophan pool size and a strong inhibition of growth rate (Table 5). DISCUSSION The value of 0.07 gmole tryptophan per g dry weight which we found is smaller than that observed for many other amino acids in yeast (Spoerl and Carleton, 1954; Stebbing, 1971), and more than ten times smaller than that reported in Neurospora crassa (Matchett et al., 1968). The only comparable measurements in yeasts were done by Weiss et al. (1975). These authors found a pool size of 1.1 nmole per 108 cells, which corresponds to approximately 0.4 gmole tryptophan per g dry weight (J. Adams, personal communication). However, the authors used the colorimetric method of Messineo and Musarra (1972) for tryptophan determination. A comparison of the tryptophanase method with the Messineo-Musarra method showed, that the colorimetric method of Messineo and Musarra gives values for the tryptophan pool which are three to four fold higher than those obtained With the tryptophanase method (J. Adams, personal communication). Taking this into consideration the data of Weiss et al. (1975) would correspond to a tryptophan pool size of around or slightly higher than 0.1 gmole per g dry weight, a value close to what we determined. From the measured tryptophan content of Saccharomyces cerevisiae protein (1.2 ~o by weight) and the growth rate p of 0.3, it is possible to calculate the necessary flux through the pathway as 0.35 nmole per minute per mg protein, assuming that all tryptophan is used for protein synthesis. The activity of anthranilate synthase measured in vitro is four to five times this value (Table 2). An estimate of the intracellular concentration of tryptophan based on our figure for gmole per dry weight is 0.02- 0.03 mM, using published conversion factors (Gancedo and Gancedo, 1973 ; Ramos et al., 1970). This concentration inhibits anthranilate synthase in vitro by 7 0 - 8 0 ~o (Schfirch et al., 1974). The flux through the pathway therefore agrees with the probable in vivo activity of the regulatory enzyme. It is uncertain whether the tryptophan pool measured by the procedure used in this work is evenly distributed throughout the cell. Yeasts are known to contain vacuoles which can act as storage reservoirs
Arch. Microbiol.,Vol. 107, No. 2 (1976) for amino acids and other molecules, and in the case of some amino acids it seems that the greater part of the pool is vacuolar (Wiemken and Nurse, 1973). Compartmentation of pools on a functional basis has also been reported (Bearden and Moses, 1972; Cowie and McClure, 1959). It seems likely that at least a substantial proportion of the tryptophan pool in wild type S. cerevisiae during growth in minimal medium is cytoplasmically located, as (i) the pool is very small (ii) the calculation presented above shows that the measured pool is of the right order to account for the inhibition of anthranilate synthase expected by comparing the in vitro activity with the flow through the pathway. In cases where the tryptophan pool is substantially higher than that of wild type cells in minimal medium, it may be that a large part of the pool is located elsewhere in the cell. The biosynthesis of tryptophan in S. cerevisiae, as in many other similar systems in fungi, appears to be controlled primarily by feedback inhibition of the first enzyme of the pathway by the end-product (Doy and Cooper, 1966; Lingens et al., 1966), and our data support this view. In some fungi, end product repression of amino acid biosynthetic pathways has been reported (Gross, 1969; De Robichon-Szulmajster and Surdin-Kerjan, 1971 ; Wiame, 1971). We observed no repression of tryptophan enzymes in the wild type strain even after addition of 200 gg tryptophan per ml, addition of precursors such as anthranilic acid or indole, or by increasing the internal tryptophan pool by introducing a feedback resistance mutation. Our results are in disagreement with those of Doy and Cooper (1966) and of Lingens et al. (1966). As the exact experimental conditions of these authors are unknown, especially with respect to growth stage at the time of harvesting, no comment can be made on the possible reason for the discrepancy. In contrast with the lack of repression, enzymes of the tryptophan pathway show a degree of derepression under some conditions. Starving a tryptophan auxotroph for tryptophan, or addition of 5-methyltryptophan can increase the enzyme activities by twoor three-fold. It is of interest to know whether derepression is caused by reduction in the tryptophan pool alone, or whether some other factor is involved. Under the derepressing conditions described, the growth rate is reduced, which led us to suspect that low growth was also required for derepression. Pool data are so far lacking for the first condition, while no reduction in pool was found in the second. Thus derepression can occur without tryptophan pool reduction provided growth is slower. A converse situation exists in strains RH424 and RH482, where growth rate is normal, the tryptophan pool is reduced, and no derepression is found.
P. A. Fantes et al. : Tryptophan Pool Size and Enzyme Levels in Saccharornyces eerevisiae
A possible link between tryptophan biosynthesis and growth rate is at the level of tryptophanyl-tRNA charging. If the charged tRNA plays an important role in regulation, then severe reduction of the tryptophan pool might affect the degree of charging and hence enzyme synthesis. The analogue 5-methyltryptophan may compete with tryptophan for the activating enzyme but does not seems to be effectively charged in Escherichia coIi (Doolittle and Yanofsky, 1968) or in yeast (Schiirch, 1972). A reduction in the degree of charging would presumably slow down protein syntheses and growth, providing the link between derepression and growth rate. An involvement of charged tryptophanyl tRNA in regulation of the synthesis of tryptophan biosynthetic enzymes has already been postulated for Neurospora crassa by Nazario et al. (1971) and by Lester (1971). This hypothesis is consistent with the observation that in continuous culture, rather severe tryptophan limitation of an auxotrophic strain is needed to bring about derepression: at a dilution rate of 0.1 h -1, corresponding to approximately one third of the maximum growth rate, derepression is only by a factor of 1.8 (T. Kieser, unpublished observations from this laboratory). Glucose limitation does not lead to derepression. Increasing the internal tryptophan pool in the presence of 5-methyltryptophan by addition of anthranilate, indole or tryptophan, or the presence of a feedback-resistant anthranilate synthase, would reduce the inhibition of charging by 5-methyltryptophan, and prevent both growth inhibition and derepression. These results are in fact observed (Gross, 1965; Schtirch et at., 1974). The presence of 5-methyltryp*;ophan does not affect the tryptophan pool in wild type cells, as this is maintained by feedback inhibition. The derepression observed in the wild type strain may also be important here, as non-derepressible mutants RH 428 and RH 487 do show a reduction in tryptophan pool under these conditions. The reduced tryptophan pool size might only permit a decreased competition with 5-methyltryptophan in the charging reaction, and these mutant strains become more sensitive to the analogue than the wild type strain. Acknowledgements. We acknowledge the competent technical assistance of Mrs. Monika Niederberger. This work was supported by the Swiss National Foundation for Scientific Research (Projects No. 3.324.70 and 3.053.73).
REFERENCES Adams, B. G.: Method for decryptification of ~-glucosidase in yeast with dimethyl sulfoxide. Analyt. Biochem. 45, 137-146 (1972)
2~3
Bearden, L., Moses, V.: Compartmentation in histidine biosynthesis. Biochim. biophys. Acta (Amst.) 279, 513- 526 (1972) Cowic, D. B., McClure, F. T. : Metabolic pools and the synthesis of macromolecules. Biochim. biophys. Acta (Amst.) 31, 236245 (1959) DeMoss, J. A. : Studies on the mechanism of the tryptophan synthetase reaction. Biochim. biophys. Acta (Amst.) 62, 279-293 (] 962) Difco Manual (Difco Laboratories Inc. Detroit, Michigan): Micro assay culture agar/twptophan assay medium, 9th ed., pp. 2 1 2 213, 235-236 (1953) Doolittle, W. F., Yanofsky, C. : Mutants of Escheric/~ia eoli with an altered tryptophanyl-transfer ribonucleic acid synthetase. J. Bact. 95, 1283-1294 (1968) Doy, C. H., Cooper, J. M. : Aromatic biosynthesis in yeast. I. The synthesis of tryptophan and the regulation of this pathway. Biochim. biophys. Acta (Amst.) 127, 302- 316 (1966) Egan, A. F., Gibson, F.: Anthranilate synthase and anthranilate5'-phosphoribosyl-l-pyrophosphate phosphoribosyl transferase (PR transferase) aggregate from Aerobacter aerogenes. In: Methods in enzymology, Vol. 17A (H. Tabor, C. W. Tabor, eds.), pp. 380- 382. London-New York: Academic Press 1970 Epstein, W., Beckwith, J. : Regulation of gene expression. Ann. Rev. Biochem. 37, 411-436 (]968) Gancedo, J. M., Gancedo, C. : Concentration of intermediary metabolites in yeast. Biochimie 55, 205-211 (1973) Gibson, F : Preparation of chorismic acid. In: Methods in enzymology, Vol. 17A (H. Tabor, C. W. Tabor, eds.), pp. 362364. New York: Academic Press 1970 Gross, S. R. : The regulation of synthesis of leucine biosynthetic enzymes in neurospora, Proc. nat. Acad. Sci. (Wash.) 54, 1538- 1546 (1965) Gross, S. R. : Genetic regulatory mechanisms in the fungi. Ann. Rev. Genet. 3, 395-424 (1969) Herbert, D., Phipps, P. J., Strange, R. E.: Chemical analysis of microbial cells. In: Methods in microbiology, Vol. 5B (J. R. Norris, D. W. Ribbons, eds.), pp. 209- 344. London-New York: Academic Press 1971 Hfitter, R.~ DeMoss, J. A.: Organisation of the tryptophan pathway: A phylogenetic study of the fungi. J. Bact. 94, 1896-1907 (1967) Kacser, H., Burns, J. A. : Causality, complexity and computer. In : Quantitative biology of metabolism, Proc. 3rd. Internat. Syrup. (A. Locker, ed.), pp. 11 - 23. Berlin-Heidelberg-New York: Springer 1968 Lester, G.: Regulation of tryptophan biosynthetic enzymes in Neurospora crassa. J. Bact. 107, 193-202 (1971) Lingens, F., Goebel, W., Uesseler, H. : Regulation der Biosynthese der aromatischen Aminosfiuren in Saccharornyces cerevisiae. I. Hemmung der Enzymaktivitfit (Feedback-Wirkung). Biochem. Z. 346, 357-367 (1966) Masselot, M., De Robichon-Sznlmajster, H. : Methionine biosynthesis in Saccharornyces cerevisiae : Mutations at the regulatory locus ETH2. II. Physiological and biochemical data. Molec. Gen. Genet. 129, 349-362 (1974) Matchett, W. H., Turner, J. R., Wiley, W. R. : The role of tryptophan in the physiology of Neurospora. Yale 3". Biol. Med. 40, 257-283 (1968) Messineo, L., Musarra, E. : A sensitive spectrophotometric method for the determination of free or bound tryptophan. Int. J. Biochem. 3, 700- 704 (1972) Nazario, M., Kinsey, J. A., Ahmad, M, : Neurospora mutant deficient in tryptophanyltransfer ribonucleic acid synthetase activity. J. Bact. 105, 121-126 (1971) Ramos, F., Thuriaux, P., Wiame, J. M., Bechet, J. : The participation of ornithine and citrulline in the regulation of arginine
214 metabolism in Saccharomyces cerevisiae. Europ. J. Biochem. 12, 4 0 - 4 7 (1970) Rasse-Messenguy, F., Fink, G. R. : Feedback-resistant mutants of histidine biosynthesis in yeast. Basic Life Sci. 2, 8 5 - 95 (1973) De Robichon-Szulmajster, H., Surdin-Kerjan, Y. : Nucleic acid and protein synthesis in yeasts: Regulation of synthesis and activity. In: The yeasts, Vol. 2 (A. H. Rose, J. S. Harrison, eds.), pp. 335-418. London-New York: Academic Press 1971 Schiirch, A. R.: Zur Regulation der Tryptophan-Biosynthese bei Saccharomyces cerevisiae. Dissertation ETHZ Nr. 4862. Ztirich: Juris 1972 Schiirch, A., Miozzari, J., Hiitter, R. : Regulation of tryptophan biosynthesis in Saccharomyces cerevisiae: Mode of action of 5-methyl-tryptophan and 5-methyl-tryptophan-sensitive mutants. J. Bact. 117, 1131-1140 (1974) Shetty, A.S., Gaertner, F. H.: Activities in microorganisms: Occurrence and properties of a single physiologically discrete enzyme in yeast. J. Bact. 113, 1127-1133 (1973) Smith, O. H., Yanofsky, C. : Enzymes involved in the biosynthesis of tryptophan. In: Methods in enzymology, Vol. 5 (S. P. Colowick, N. O. Kaplan, eds.), pp. 794-806. London-New York: Academic Press 1962
Arch. Microbiol., Vol. 107, No. 2 (1976) Spoerl, E., Carleton, R. : Studies on cell division. Nitrogen compound changes in yeast accompanying an inhibition of cell division. J. biol. Chem. 210, 521-529 (1954) Stebbing, N. : Growth and changes in pool and macromolecular components of Schizosaccharomyees pornbe during the cell cycle. J. Cell Sci. 9, 701-717 (1971) Weiss, R. L., Kukora, J. R., Adams, J. : The relationship between enzyme activity, cell geometry, and fitness in Saccharornyees cerevisiae. Proc. nat. Acad. Sci. (Wash.) 72, 794-798 (1975) Wiame, J. M. : Mechanism of the interaction between anabolism and catabolism of arginine in Saccharomyces cerevisiae. In: Recent advances in microbiology (A. P6rez-Miravete, D. Pelaez, eds.), pp. 243-253. Mexico, D.F.: Xth International Congress for Microbiology 1971 Wiemken, A., Nurse, P. : The vacuole as a compartment of amino acid pools in yeast. In: Proc. 3rd. Int. Symp. on Yeast, Part II (H. Suomalainen, C. Waller, eds.), pp. 331- 347. Helsinki: Print Oy 1973
Received October 27, 1975
Microbiology
Arch. Microbiol. 107, 207-214 (1976)
9 by Springer-Verlag 1976
Free Tryptophan Pool and Tryptophan Biosynthetic Enzymes in Saccharomyces cerevisiae PETER A. FANTES*, LILIAN M. ROBERTS, and RALF HUETTER Mikrobiologisches Institut der Eidgen6ssischen Technischen Hochschule, Universit/itsstr. 2, CH-8006 Ziirich, Switzerland
Abstract. The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties. The tryptophan pool of wild type cells growing in minimal medium is 0.07 gmole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth" medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool. Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by L-tryptophan or by the analogue DL-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced ~ryptophan pool, but no derepression of indoleglycerolphosphate synthase was found. A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.
* Present address: Department of Zoology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JN, Scotland. Abbreviations. CDRP = 1-(o-carboxyphenylamino)-l-deoxyribulosephosphate; paba = paraaminobenzoic acid; PRA = N(5'-phosphoribosyl)-anthranilate; tRNA = transfer ribonucleic acid; trpl to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes.
Key words." Anthranilate synthase - Cell permeabilisation - Indoleglycerolphosphate synthase - Saccharomyces cerevisiae - Tryptophan biosynthetic enzymes - Tryptophan pool.
A substantial body of information now exists about the regulation of biosynthetic pathways in yeast and other fungi (De Robichon-Szulmajster and SurdinKerjan, 1971; Gross, 1969). Much of the published work describes the variation in enzyme levels under different nutritional conditions, and the effect of regulatory mutations on them. In only a few cases, however, have the internal concentrations of intermediates and the end product of the pathway been measured (Masselot and De Robichon-Szulmajster, 1974; Ramos et al., 1970; Rasse-Messenguy and Fink, 1973). This report is concerned with the interactions between the tryptophan pool and tryptophan biosynthetic pathway, using different conditions of growth and mutants with altered regulatory properties. A simplified scheme of tryptophan biosynthesis is given in Figure 1. The great majority of tryptophan is probably used for protein synthesis, as (i) the amount needed for nicotinic acid biosynthesis is very small, and this vitamin is present in the medium, (ii) exogenous tryptophan is used only slowly as nitrogen source, indicating a low rate of tryptophan degradation in Saccharomyces cerevisiae (Shetty and Gaertner, 1973). The enzymes assayed in this work, anthranilate synthase and indoleglycerolphosphate synthase, first and fourth enzymes respectively of the tryptophan pathway, are known to occur as an aggregate in vitro, and are thought to do so in whole cells (Htitter and DeMoss, 1967). As their regulation appears to be coordinate (Schiirch et al., 1974), we are able to assay the aggregate in mutants with altered anthranilate synthase by measuring indoleglycerolphosphate synthase.
208
Arch. Microbiol., Vol. 107, No. 2 (1976) REPRESSION
f
INHIBITION
Chorismate 9
~. Anthranilate
~ PRA
~ CDRP :
phe, tyr
paba
anthranilate synthase~ trp 2, tr_~p_3
. ~ Indoleglycerol : phosphate i
anthranilate phosphori- indoleglycerolphosphorihosylbosyl phosphate transferase anothg2n~I2te synthase " ~ 4
~
p
3
tryptophan synthase
Tryptophan I I I
NAD
~ Tryptophanyl-tRNA
1
Protein
trp 5
enzyme aggregate Fig. 1. Tryptophan biosynthesis in Saccharomyees cerevisiae
F r o m p r e v i o u s studies it is k n o w n t h a t starving a t r y p t o p h a n a u x o t r o p h for t r y p t o p h a n leads to derep r e s s i o n ( M i o z z a r i , J., u n p u b l i s h e d d a t a f r o m this labor a t o r y ) a n d t h a t the t r y p t o p h a n a n a l o g u e DL-5-methylt r y p t o p h a n causes a r e d u c t i o n in g r o w t h r a t e a n d a d e r e p r e s s i o n o f the t w o e n z y m e s a n t h r a n i l a t e synthase a n d i n d o l e g l y c e r o l p h o s p h a t e synthase b y a f a c t o r o f a b o u t 2.5 (Schtirch et al., 1974). T o gain a b e t t e r insight into the r e g u l a t o r y process, the t r y p t o p h a n p o o l a n d e n z y m e activities have been m e a s u r e d here in e x p o nentially g r o w i n g cells, a n d c o n d i t i o n s w h e r e g r o w t h is u n b a l a n c e d , such as s t a r v a t i o n o f a t r y p t o p h a n a u x o t r o p h for t r y p t o p h a n , have been a v o i d e d . R e d u c tion in the t r y p t o p h a n a v a i l a b l e to the Cells was b r o u g h t a b o u t b y using m u t a n t s with low e n z y m e activity, a l o n g the lines o f the " m o d u l a t i o n " experim e n t s o f K a c s e r a n d Burns (1968). I n a d d i t i o n to the wild t y p e strain, two classes o f r e g u l a t o r y m u t a n t s have been s t u d i e d in o u r laboratory: those showing altered feedback parameters o f a n t h r a n i l a t e s y n t h a s e (Schtirch et al., 1974), a n d those w h i c h h a v e lost the ability t o d e r e p r e s s (Schiirch et al., 1974) or repress the synthesis o f at least the t w o t r y p t o p h a n enzymes a s s a y e d here.
pH adjusted to 4.0 with KOH. The use of buffered medium reduces variation in the inhibition of growth rate by 5-methyltryptophan. Media and supplements except 5-methyltryptophan were sterilised by autoclaving separately at 120~ for 20 min; 5-methyltryptophan was filter sterilised. Concentrations of 5-methyltryptophan added to the growth media are indicated in the text. Cultures were grown at 30~C, liquid cultures on a rotary shaker at 150 strokes per minute. Growth was followed turbidimetrically at 546 nm with a 1 cm light path, and dry weights related to this using a calibration curve. The specific growth rate, #, is defined as = (ln x2-1n Xt)/(t2-tl). One unit of optical density (OD546 of 1.0) corresponds to approx. 1.5 9 10l~ cells per ml, which equal 1.1 g wet weight, 165 mg dry weight or 80 mg protein.
MATERIALS
Enzyme Assays 1. Anthranilate synthase activity was determined using a "stop assay" (Egan and Gibson, 1970). The reaction was started by adding 0.2 ml of permeabilised cell suspension suitably diluted in KMG buffer to the assay mix, which contained, in a final volume of 1.0 ml: 0.1 mmole potassium phosphate, pH 7.6; 5 ixmole MgSO4; 20 gmole L-glutamine; 0.1 gmole chorismic acid. The assay was run for 20 rain at 37~ and stopped by adding 0.1 ml i N HC1. The product, anthranilic acid, was extracted in 4 ml ethyl
AND METHODS
Chemicals. The tryptophan analogueDL-5-methyltryptophan (puriss. grade) was obtained from Fluka AG, Buchs SG, Switzerland. Escherichia coli tryptophanase (grade II, lot 43C-6910, activity quoted per mg: 30 pg indole released from L-tryptophan per 10 min at pH 8.3 and 37~ was purchased from Sigma Chemical Co., St. Louis, Missouri. Chorismic acid was made according to Gibson (1970), and CDRP [1-(o-carboxyphenylamino)-l-deoxyribulose phosphate) was made by the method of Smith and Yanofsky (1962). Other chemicals were of analytical grade. Media and Growth Conditions. YEPD medium (1 ~ oxoid yeast extract, 2 ~ oxoid peptone, 2 ~ glucose plus 2 ~ agar for solid medium) was used as complete medium and for maintenance of strains. The minimal medium used was MV medium (0.145 ~o Difco yeast nitrogen base without amino acids and ammonium sulphate, 0.525 G ammonium sulphate, 2 ~ glucose) with 1 G succinic acid,
Permeabilisation of Cellsfor Enzyme Assays. Cultures were harvested at an optical density of approximately 1.0 by centrifugation at 2-4~ After resuspension in "KMG" buffer (0.1 M potassium phosphate, pH 7.6, containing 5 mM magnesium sulphate and 20 mM L-glutamine as stabilisers for anthranilate synthase), the cells were again centrifuged, and resuspended to 100 mg wet weight per ml in KMG. Dimethylsulphoxide was added to a final concentration of 40~ (v/v) and after standing for 10 min at 0~ the cells were centrifuged and resuspended in KMG. The washing was repeated and the final resuspension made in KMG to 100 mg wet weight per ml (Adams, 1972). Enzyme activities were stable for at least 6 h under these conditions. Protein was estimated in the permeabilised cell preparations by the biuret method described by Herbert et al. (1971). Control experiments showed that the permeabilisation has no detectable effect on the protein content of the cells.
t Anthranilate synthase [chorismate pyrt/vate lyase (amino accepting) ; EC 4.1.3.27] refers to the activity measured with glutamine as amino donor; anthranilate phosphoribosyltransferase [N-(5'phosphoribosyl)-anthranilate pyrophosphate phosphoribosyltransferase; EC 2.4.2.18]; indoleglycerolphosphate synthase [1-(2'-carboxyphenylamino)- 1-deoxyribulose- 5-phosphate carboxylase-[cyclizing); EC 4.t.1.48]; phosphoribosyl-anthranilate isomerase [N(5'-phosphoribosyl)-anthranilate isomerase]; tryptophan synthase [L-serine hydrolyase (adding indole); EC 4.2.1.20].
P. A. Fantes et al. : Tryptophan Pool Size and Enzyme Levels in Saccharomycescerevisiae
209
Table 1. Strains of Saecharomyees cerevisiae Strain
Growth rate in MV medium
~ Growth inhibition by 5 • 10 -4 M DL-5methyltryptophan (liquid culture)
Growth sensitivity to 10 -~ M DL-5fluortryptophan (solid medium)
Description (other phenotypic characters)
X2180-1A
0.310
29
Sensitive
Wild type strain
RH 424
0.272
50
Sensitive
Anthranilate synthase reduced and feedback-supersensitive (maps in trp2) (Sch/ireh et al., 1974)
RH 428 RH 482
0.268 0.276
47 52
Sensitive Sensitive
RH 487 RH 595
0,265 0.291 "
65 0
Sensitive Resistant
Non-derepressible for tryptophan enzymes (Schtirch et al., 1974) Anthranilate synthase greatly reduced and feedback-supersensitive (maps in trp2) (Schfirch et al., 1974) Non-derepressible for tryptophan enzymes (Schiirch et al., 1974) Slightly feedback-resistant anthranilate synthase; derepressed enzyme levels
RH 596
0.257
20
Sensitive
RH 597 RH 598
0.281 0.233
0 0
Resistant Resistant
acetate, and estimated by its fluorescence measured at 340 nm excitation, 408 nm emission, on an Aminco-Bowman Spectrofiuorimeter (Egan and Gibson, 1970). Indoleglycerolphosphate synthase was assayed as described previously (Schtirch et al., 1974), using permeabilised cells instead of cell-free extract.
Extraction of Soluble Pool of Cells. For most experiments, four parallel cultures of 400 ml MV medium plus supplements were used for each experimental condition. The cultures were harvested by vacuum filtration on Whatman GF/C fiiters supported in Millipore 47 mm filtration units, and washed twice with 25 ml ice-cold water. Filters were removed and all four immersed in 10 ml icecold (60 ~ (v/v) ethanol. After 15 rain, a portion of the extracted suspension was filtered through a GF/C filter and washed with 10 ml 60~o ethanol. The filtrate was stored at - 2 5 ~ Extracts were evaporated to dryness on a rotary evaporator at 35-40~ and the material taken up in a small volume (1 - 5 ml) of water.
Derepressed enzyme levels; derived from RH 595 by recombination Feedback-resistant anthranilate synthase (maps in trp2) Feedback-resistant anthranilate synthase (maps in trp2)
(up to 50-fold) of 5-methyltryptophan. Three different sample volumes were assayed for each determination. Media were obtained from Difco, and the organism used was Lactobacillus plantarum, ATCC 8014.
Strains of Saccharomyces cerevisiae. All the strains used were derived by mutation and recombination from the isogenic wild type strains X2180-1A (a mating type) and X2180-1B (c~ mating type). The main characteristics of the strains are presented in Table 1. Strain RH 595 was isolated after N-methyl-N'-nitro-N-nitrosoguanidine treatment of the wild type strain X2180-1A as a 5-fluortryptophan resistant mutant. Strain RH 597 was isolated as a spontaneous 5-fluor-tryptophan resistant mutant from the same wild type strain. Strain RH 598 was isolated as a feedback resistant segregant from strain RH 446, a 5-methyltryptophan resistant revertant of strain RH 487.
Estimation of Tryptophan. Two methods were used, which gave
RESULTS
results within 20 ~ of one another. (i) Enzymatic assay (DeMoss, 1962). This method was the more accurate of the two, but less sensitive. It couId not be used to determine tryptophan in the presence of 5-methyltryptophan, as the latter interferes. A sample of the material to be assayed was incubated with tryptophanase in a buffer system with the following composition in a final volume of 1.2 ml: 0.144 mmole potassium phosphate, pH 7.8; 0.24 gmole dithiothreitol; 0.072 gmole pyridoxal phosphate; 0.2 mg tryptophanase preparation. The test sample was overlaid with 1.0 ml toluene. Assay tubes were stoppered and incubated in a shaking water bath at 37~ for 2h. Then 0.2ml 1 N NaOH was added to each tube, and 0.5 ml of the toluene layer assayed for the reaction product, indole, by the :method of Smith and Yanofsky (1962). Indole and tryptophan standards were included in each set of assays. Recovery of known amounts of L-tryptophan was between 90 and 100 ~. (ii) Microbiological assay. The procedure followed is based on that described in the Difco Manual (1953). The main difference is that growth was measured turbidimetrically between 12 and 15 h after inoculation, rather than acidimetrically. This procedure made it possible to assay tryptophan in the presence of a large excess
Tryptophan Pool in the Wild Type Strain A s s h o w n in T a b l e s 2 - 5 , t h e t r y p t o p h a n p o o l in w i l d t y p e cells g r o w i n g in m i n i m a l m e d i u m is n e a r 0.07 g m o l e p e r g d r y w e i g h t . T h e a d d i t i o n o f t r y p t o p h a n o r m e t a b o l i c p r e c u r s o r s to t h e m e d i u m b y p a s s e s the control by feedback inhibition of anthranilate s y n t h a s e , a n d t h e t r y p t o p h a n p o o l is g r e a t l y i n c r e a s e d u n d e r t h e s e c o n d i t i o n s ( T a b l e 2). T h e p r e s e n c e o f s u c h a h i g h t r y p t o p h a n p o o l d i d n o t , h o w e v e r , l e a d to repression of the tryptophan enzymes.
Tryptophan Pool and Enzyme Activities in Mutants with Altered Feedback Properties of Anthranilate Synthase T w o classes o f m u t a n t s w i t h a l t e r e d f e e d b a c k p r o p e r ties in a n t h r a n i l a t e s y n t h a s e w e r e a n a l y s e d , f e e d b a c k
210
Arch. Microbiol., Vot. 107, No. 2 (1976)
Table 2. Effectof growth medium on tryptophan pools and enzyme activities in wild type strain X2180-1A Growth medium
Growth rate
MV MV + Tryptophan ~ MV + Indol& MV + Anthranilic acid"
0.310 0.310 0.307 0.279
Specific enzyme activities (nmole mg protein- 1 min -1) Anthranilate synthase
Indoleglycerolphosphate synthase
1.40 1.10 1.38 1.20
2.15 2.33 2.60 1.85
Tryptophan pool (lamole g dry weight-i)
0.062 0.708 3.40 1.80
Supplements added at 20 lag m1-1. Table 3. Tryptophan pools and enzyme activities in mutants with altered feedback sensitivity Strain
X2180-1A RH 597 RH 598 RH 424 RH 482
Growth rate in MV medium
0.309 0.281 0.233 0.272 0.276
Specific enzyme activities (nmole mg protein-t min- i) Anthranilate synthase
Indoleglycerolphosphate synthase
1.32 1.42 1.38 0.33 0.05
2.23 2.77 1.83 1.62 2.62
Feedback sensitivity"
Tryptophan pool (pmole g dry weight-1)
10-5 > 10.3 > 10-3 2x 10 6 3 x 10-6
0.065 3.75 3.12 0.036 0.037
Tryptophan pools less than 0.1 lamole per g dry weight were determined a minimum of two times. Values given are means. a Concentration of L-tryptophan which inhibits the activity by 50 ~ under the conditions described in "Materials and Methods". Table 4. Tryptophan pool and enzyme activities in regulatory mutants Strain
X2180-1A RH 595 RH 596 RH 487 RH 428
Growth rate in MV medium
0.281 0.291 0.257 0.265 0.268
Specific enzyme activities (nmole mg protein-1 min 1) Anthranilate synthase
Indoleglycerolphosphate synthase
1.32 3.03 2.65 1.07 1.00
2.25 4.42 3.63 1.38 1.20
Tryptophan pool (pmole g dry weight -t)
0.072 2.70 1.22 0.095 0.069
Tryptophan pools less than 0.1 pmole per g dry weight were determined a minium of two times. Values given are means.
resistant and feedback supersensitive strains. It was possible that although externally supplied t r y p t o p h a n or its metabolic precursors w o u l d n o t repress the biosynthetic p a t h w a y , internally synthesised t r y p t o p h a n might do so: differential effects o f externally supplied and endogenously synthesised t r y p t o p h a n have been r e p o r t e d in Neurospora crassa (Matchett et al., 1968), and yeasts m a y contain a m i n o acids in several physiologically distinct pools (Bearden and Moses, 1972; Cowie and McClure, 1959). To test this possibility, we m a d e use o f m u t a n t s with altered anthranilate synthase which is less sensitive to inhibition by tryptop h a n than the wild type strain. Table 3 shows that
the t r y p t o p h a n p o o l in these m u t a n t s is very high, higher than that obtained in the wild type strain under m o s t conditions. The activity o f indoleglycerolphosphate synthase is however no lower than in the wild type strain. These experiments show that if t r y p t o p h a n itself is the molecule corresponding to the corepressor in bacterial systems (Epstein and Beckwith, 1968), then the t r y p t o p h a n biosynthetic p a t h w a y in Saccharomyces cerevisiae is fully repressed during growth on minimal medium. I f t r y p t o p h a n pool size is a controlling factor in the regulation o f its o w n biosynthesis at the level o f
P. A. Fantes et al. : Tryptophan Pool Size and Enzyme Levels in Saccharomyces cerevisiae
211
Table 5. Effect of 5-methyltryptophan on tryptophan pools and enzyme levels in wild type strain X2180-1A and the mutant strains RH 428 and RH 487 Growth medium
Growth rate
Specific enzyme activity (nmole mg protein- ~ min-1) Anthranilate synthase
Indoleglycerolphosphate synthase
Tryptophan pool (gmole g dry weight -1) (mean values and standard error)
Wild type strain : MV MV + DL-5-methyltryptophan (5 x 10 -4 M)
0.295 0.194
1.40 3.22
2.15 6.50
0.069 • 0.004 0.073 _+ 0.011
Mutant strain RH 428 : MV MV + DC-5-methyltryptophan (10 .4 M)
0.296 0.067
1.48 0.85
1.70 1.12
0.068 ___ 0.002 0.039
Mutant strain RH 487: MV MV + DL-5-methyltryptophan (10 .4 M)
0.264 0.073
1.16 1.36
1.50 1.76
0.11 0.05
Where standard errors are given the values for the tryptophan pool are based on ten determinations (both enzymatic and microbiological) for MV medium, and four determinations (microbiological only) for MV + 5 MT medium.
enzyme synthesis, then a reduction in pool size should lead to derepression of the pathway. To test this, we used mutants with decreased anthranilate synthase activity which was also more sensitive to inhibition by tryptophan than the enzyme of the wild type strain. Table 3 shows that the pool in these mutants is reduced by about one half, which is a smaller decrease than the 75 ~ and 96 ~ reduction in anthranilate synthase activity in strains RH 424 and R H 482 respectively. Despite the smaller tryptophan pools in these mutants, there is no evidence for derepression of indoleglycerolphosphate synthase in either. It should be pointed out that both strains have exponential growth rates essentially the same as the wild type strain, in spite of their low biosynthetic capacity.
Tryptophan Pool and Enzyme Activity in Mutants with Altered Regulation of Tryptophan Biosynthetic Enzymes Two types of mutants of this class have been isolated. One type of regulatory mutant has only recently been isolated, which maintains higher enzyme levels than the wild type strain under all growth conditions tested and may therefore be considered "derepressed". The main characteristics are summarised in Table i. Pools and enzyme levels were determined for strains R H 595 and R H 596, and the results in Table 4 show an increase in the two enzyme activities and the pool size. This shows that the high tryptophan pool is a result of the increased enzyme levels, rather than the enzyme levels being high as a result of tryptophan starvation. Strain R H 595 has a higher pool than strain R H 596 pre-
sumably as a consequence of the relative insensitivity of its anthranilate synthase to tryptophan inhibition. Mutants of the other type have been described previously (Schtirch et al., 1974). They have lost the ability to derepress the biosynthetic enzymes in response to either the presence of 5-methyltryptophan or deprivation of tryptophan when combined with tryptophan auxotrophy in a double mutant. The results in Table 4 show that the inability to derepress is not due to a permanently high internal tryptophan pool.
Effect of 5-Methyltryptophan on Tryptophan Pool and Enzyme Activities As described in a previous report (Schfirch et al., 1974), inclusion of DL-5-methyltryptophan in the growth medium produces an inhibition of growth of the wild type strain which under the conditions described here is about 30 ~, and leads to a derepression of the biosynthetic enzymes of about 2.5-fold (Table 5). The analogue inhibits anthranilate synthase in extracts and permeabilised cells to about the same degree as tryptophan, and these results led Schfirch et al. (1974) to the following hypothesis: 5-methyltryptophan inhibits anthranilate synthase and causes a reduction in tryptophan biosynthesis, which leads to a reduced tryptophan pool. The reduction in pool size then effects a derepression of the biosynthetic pathway. To test this hypothesis more directly, the tryptophan pool in cells growing in the presence of the analogue was determined. The results (Table 5) show that there is little, if any difference between pool size of wild type cells grown in the presence or absence of this
212 analogue. Any reduction in the tryptophan pool caused by the analogue is smaller than that produced by a defective anthranilate synthase (see Table 3). For mutants lacking the ability to derepress the tryptophan biosynthetic enzymes in response to 5-methyltryptophan, strains RH 428 and RH 487, the situation is different. In this case 5-methyltryptophan causes a significant reduction in tryptophan pool size and a strong inhibition of growth rate (Table 5). DISCUSSION The value of 0.07 gmole tryptophan per g dry weight which we found is smaller than that observed for many other amino acids in yeast (Spoerl and Carleton, 1954; Stebbing, 1971), and more than ten times smaller than that reported in Neurospora crassa (Matchett et al., 1968). The only comparable measurements in yeasts were done by Weiss et al. (1975). These authors found a pool size of 1.1 nmole per 108 cells, which corresponds to approximately 0.4 gmole tryptophan per g dry weight (J. Adams, personal communication). However, the authors used the colorimetric method of Messineo and Musarra (1972) for tryptophan determination. A comparison of the tryptophanase method with the Messineo-Musarra method showed, that the colorimetric method of Messineo and Musarra gives values for the tryptophan pool which are three to four fold higher than those obtained With the tryptophanase method (J. Adams, personal communication). Taking this into consideration the data of Weiss et al. (1975) would correspond to a tryptophan pool size of around or slightly higher than 0.1 gmole per g dry weight, a value close to what we determined. From the measured tryptophan content of Saccharomyces cerevisiae protein (1.2 ~o by weight) and the growth rate p of 0.3, it is possible to calculate the necessary flux through the pathway as 0.35 nmole per minute per mg protein, assuming that all tryptophan is used for protein synthesis. The activity of anthranilate synthase measured in vitro is four to five times this value (Table 2). An estimate of the intracellular concentration of tryptophan based on our figure for gmole per dry weight is 0.02- 0.03 mM, using published conversion factors (Gancedo and Gancedo, 1973 ; Ramos et al., 1970). This concentration inhibits anthranilate synthase in vitro by 7 0 - 8 0 ~o (Schfirch et al., 1974). The flux through the pathway therefore agrees with the probable in vivo activity of the regulatory enzyme. It is uncertain whether the tryptophan pool measured by the procedure used in this work is evenly distributed throughout the cell. Yeasts are known to contain vacuoles which can act as storage reservoirs
Arch. Microbiol.,Vol. 107, No. 2 (1976) for amino acids and other molecules, and in the case of some amino acids it seems that the greater part of the pool is vacuolar (Wiemken and Nurse, 1973). Compartmentation of pools on a functional basis has also been reported (Bearden and Moses, 1972; Cowie and McClure, 1959). It seems likely that at least a substantial proportion of the tryptophan pool in wild type S. cerevisiae during growth in minimal medium is cytoplasmically located, as (i) the pool is very small (ii) the calculation presented above shows that the measured pool is of the right order to account for the inhibition of anthranilate synthase expected by comparing the in vitro activity with the flow through the pathway. In cases where the tryptophan pool is substantially higher than that of wild type cells in minimal medium, it may be that a large part of the pool is located elsewhere in the cell. The biosynthesis of tryptophan in S. cerevisiae, as in many other similar systems in fungi, appears to be controlled primarily by feedback inhibition of the first enzyme of the pathway by the end-product (Doy and Cooper, 1966; Lingens et al., 1966), and our data support this view. In some fungi, end product repression of amino acid biosynthetic pathways has been reported (Gross, 1969; De Robichon-Szulmajster and Surdin-Kerjan, 1971 ; Wiame, 1971). We observed no repression of tryptophan enzymes in the wild type strain even after addition of 200 gg tryptophan per ml, addition of precursors such as anthranilic acid or indole, or by increasing the internal tryptophan pool by introducing a feedback resistance mutation. Our results are in disagreement with those of Doy and Cooper (1966) and of Lingens et al. (1966). As the exact experimental conditions of these authors are unknown, especially with respect to growth stage at the time of harvesting, no comment can be made on the possible reason for the discrepancy. In contrast with the lack of repression, enzymes of the tryptophan pathway show a degree of derepression under some conditions. Starving a tryptophan auxotroph for tryptophan, or addition of 5-methyltryptophan can increase the enzyme activities by twoor three-fold. It is of interest to know whether derepression is caused by reduction in the tryptophan pool alone, or whether some other factor is involved. Under the derepressing conditions described, the growth rate is reduced, which led us to suspect that low growth was also required for derepression. Pool data are so far lacking for the first condition, while no reduction in pool was found in the second. Thus derepression can occur without tryptophan pool reduction provided growth is slower. A converse situation exists in strains RH424 and RH482, where growth rate is normal, the tryptophan pool is reduced, and no derepression is found.
P. A. Fantes et al. : Tryptophan Pool Size and Enzyme Levels in Saccharornyces eerevisiae
A possible link between tryptophan biosynthesis and growth rate is at the level of tryptophanyl-tRNA charging. If the charged tRNA plays an important role in regulation, then severe reduction of the tryptophan pool might affect the degree of charging and hence enzyme synthesis. The analogue 5-methyltryptophan may compete with tryptophan for the activating enzyme but does not seems to be effectively charged in Escherichia coIi (Doolittle and Yanofsky, 1968) or in yeast (Schiirch, 1972). A reduction in the degree of charging would presumably slow down protein syntheses and growth, providing the link between derepression and growth rate. An involvement of charged tryptophanyl tRNA in regulation of the synthesis of tryptophan biosynthetic enzymes has already been postulated for Neurospora crassa by Nazario et al. (1971) and by Lester (1971). This hypothesis is consistent with the observation that in continuous culture, rather severe tryptophan limitation of an auxotrophic strain is needed to bring about derepression: at a dilution rate of 0.1 h -1, corresponding to approximately one third of the maximum growth rate, derepression is only by a factor of 1.8 (T. Kieser, unpublished observations from this laboratory). Glucose limitation does not lead to derepression. Increasing the internal tryptophan pool in the presence of 5-methyltryptophan by addition of anthranilate, indole or tryptophan, or the presence of a feedback-resistant anthranilate synthase, would reduce the inhibition of charging by 5-methyltryptophan, and prevent both growth inhibition and derepression. These results are in fact observed (Gross, 1965; Schtirch et at., 1974). The presence of 5-methyltryp*;ophan does not affect the tryptophan pool in wild type cells, as this is maintained by feedback inhibition. The derepression observed in the wild type strain may also be important here, as non-derepressible mutants RH 428 and RH 487 do show a reduction in tryptophan pool under these conditions. The reduced tryptophan pool size might only permit a decreased competition with 5-methyltryptophan in the charging reaction, and these mutant strains become more sensitive to the analogue than the wild type strain. Acknowledgements. We acknowledge the competent technical assistance of Mrs. Monika Niederberger. This work was supported by the Swiss National Foundation for Scientific Research (Projects No. 3.324.70 and 3.053.73).
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2~3
Bearden, L., Moses, V.: Compartmentation in histidine biosynthesis. Biochim. biophys. Acta (Amst.) 279, 513- 526 (1972) Cowic, D. B., McClure, F. T. : Metabolic pools and the synthesis of macromolecules. Biochim. biophys. Acta (Amst.) 31, 236245 (1959) DeMoss, J. A. : Studies on the mechanism of the tryptophan synthetase reaction. Biochim. biophys. Acta (Amst.) 62, 279-293 (] 962) Difco Manual (Difco Laboratories Inc. Detroit, Michigan): Micro assay culture agar/twptophan assay medium, 9th ed., pp. 2 1 2 213, 235-236 (1953) Doolittle, W. F., Yanofsky, C. : Mutants of Escheric/~ia eoli with an altered tryptophanyl-transfer ribonucleic acid synthetase. J. Bact. 95, 1283-1294 (1968) Doy, C. H., Cooper, J. M. : Aromatic biosynthesis in yeast. I. The synthesis of tryptophan and the regulation of this pathway. Biochim. biophys. Acta (Amst.) 127, 302- 316 (1966) Egan, A. F., Gibson, F.: Anthranilate synthase and anthranilate5'-phosphoribosyl-l-pyrophosphate phosphoribosyl transferase (PR transferase) aggregate from Aerobacter aerogenes. In: Methods in enzymology, Vol. 17A (H. Tabor, C. W. Tabor, eds.), pp. 380- 382. London-New York: Academic Press 1970 Epstein, W., Beckwith, J. : Regulation of gene expression. Ann. Rev. Biochem. 37, 411-436 (]968) Gancedo, J. M., Gancedo, C. : Concentration of intermediary metabolites in yeast. Biochimie 55, 205-211 (1973) Gibson, F : Preparation of chorismic acid. In: Methods in enzymology, Vol. 17A (H. Tabor, C. W. Tabor, eds.), pp. 362364. New York: Academic Press 1970 Gross, S. R. : The regulation of synthesis of leucine biosynthetic enzymes in neurospora, Proc. nat. Acad. Sci. (Wash.) 54, 1538- 1546 (1965) Gross, S. R. : Genetic regulatory mechanisms in the fungi. Ann. Rev. Genet. 3, 395-424 (1969) Herbert, D., Phipps, P. J., Strange, R. E.: Chemical analysis of microbial cells. In: Methods in microbiology, Vol. 5B (J. R. Norris, D. W. Ribbons, eds.), pp. 209- 344. London-New York: Academic Press 1971 Hfitter, R.~ DeMoss, J. A.: Organisation of the tryptophan pathway: A phylogenetic study of the fungi. J. Bact. 94, 1896-1907 (1967) Kacser, H., Burns, J. A. : Causality, complexity and computer. In : Quantitative biology of metabolism, Proc. 3rd. Internat. Syrup. (A. Locker, ed.), pp. 11 - 23. Berlin-Heidelberg-New York: Springer 1968 Lester, G.: Regulation of tryptophan biosynthetic enzymes in Neurospora crassa. J. Bact. 107, 193-202 (1971) Lingens, F., Goebel, W., Uesseler, H. : Regulation der Biosynthese der aromatischen Aminosfiuren in Saccharornyces cerevisiae. I. Hemmung der Enzymaktivitfit (Feedback-Wirkung). Biochem. Z. 346, 357-367 (1966) Masselot, M., De Robichon-Sznlmajster, H. : Methionine biosynthesis in Saccharornyces cerevisiae : Mutations at the regulatory locus ETH2. II. Physiological and biochemical data. Molec. Gen. Genet. 129, 349-362 (1974) Matchett, W. H., Turner, J. R., Wiley, W. R. : The role of tryptophan in the physiology of Neurospora. Yale 3". Biol. Med. 40, 257-283 (1968) Messineo, L., Musarra, E. : A sensitive spectrophotometric method for the determination of free or bound tryptophan. Int. J. Biochem. 3, 700- 704 (1972) Nazario, M., Kinsey, J. A., Ahmad, M, : Neurospora mutant deficient in tryptophanyltransfer ribonucleic acid synthetase activity. J. Bact. 105, 121-126 (1971) Ramos, F., Thuriaux, P., Wiame, J. M., Bechet, J. : The participation of ornithine and citrulline in the regulation of arginine
214 metabolism in Saccharomyces cerevisiae. Europ. J. Biochem. 12, 4 0 - 4 7 (1970) Rasse-Messenguy, F., Fink, G. R. : Feedback-resistant mutants of histidine biosynthesis in yeast. Basic Life Sci. 2, 8 5 - 95 (1973) De Robichon-Szulmajster, H., Surdin-Kerjan, Y. : Nucleic acid and protein synthesis in yeasts: Regulation of synthesis and activity. In: The yeasts, Vol. 2 (A. H. Rose, J. S. Harrison, eds.), pp. 335-418. London-New York: Academic Press 1971 Schiirch, A. R.: Zur Regulation der Tryptophan-Biosynthese bei Saccharomyces cerevisiae. Dissertation ETHZ Nr. 4862. Ztirich: Juris 1972 Schiirch, A., Miozzari, J., Hiitter, R. : Regulation of tryptophan biosynthesis in Saccharomyces cerevisiae: Mode of action of 5-methyl-tryptophan and 5-methyl-tryptophan-sensitive mutants. J. Bact. 117, 1131-1140 (1974) Shetty, A.S., Gaertner, F. H.: Activities in microorganisms: Occurrence and properties of a single physiologically discrete enzyme in yeast. J. Bact. 113, 1127-1133 (1973) Smith, O. H., Yanofsky, C. : Enzymes involved in the biosynthesis of tryptophan. In: Methods in enzymology, Vol. 5 (S. P. Colowick, N. O. Kaplan, eds.), pp. 794-806. London-New York: Academic Press 1962
Arch. Microbiol., Vol. 107, No. 2 (1976) Spoerl, E., Carleton, R. : Studies on cell division. Nitrogen compound changes in yeast accompanying an inhibition of cell division. J. biol. Chem. 210, 521-529 (1954) Stebbing, N. : Growth and changes in pool and macromolecular components of Schizosaccharomyees pornbe during the cell cycle. J. Cell Sci. 9, 701-717 (1971) Weiss, R. L., Kukora, J. R., Adams, J. : The relationship between enzyme activity, cell geometry, and fitness in Saccharornyees cerevisiae. Proc. nat. Acad. Sci. (Wash.) 72, 794-798 (1975) Wiame, J. M. : Mechanism of the interaction between anabolism and catabolism of arginine in Saccharomyces cerevisiae. In: Recent advances in microbiology (A. P6rez-Miravete, D. Pelaez, eds.), pp. 243-253. Mexico, D.F.: Xth International Congress for Microbiology 1971 Wiemken, A., Nurse, P. : The vacuole as a compartment of amino acid pools in yeast. In: Proc. 3rd. Int. Symp. on Yeast, Part II (H. Suomalainen, C. Waller, eds.), pp. 331- 347. Helsinki: Print Oy 1973
Received October 27, 1975
