Synthesis and Activation of Asparagine in Asparagine Auxotrophs of Saccharomyces cerevisiae

Eur. J . Biochem. 94, 409-417 (1979)

Synthesis and Activation of Asparagine in Asparagine Auxotrophs of Saccharomyces cerevisiae

Fernando RAMOS and Jean-Marie WIAME

Laboratoire de Microbiologie, Facultt des Sciences, Universitt Libre de Bruxelles, and Institut de Recherches du Centre d’Enseignement et de Recherches des Industries Alimentaires et Chimique, Bruxelles

(Received October 19, 1978)

L-Asparagine synthesis in Saccharomyces cerevisiae is performed by a glutamine-dependent asparagine synthetase of the type found in higher organisms.

Auxotrophy for asparagine has been obtained in two classes of mutants. In class I, asparagine synthetase activity is cancelled. These mutants combine two mutations, asnA - and asnB- . Neither asnA- nor asnB- mutation alone leads to total auxotrophy. Partial auxotrophy as well as a strong decrease in enzyme activity result from asnA- mutation. No change is detectable in cells with the asnB- mutation alone. This, and Jones’ report [ J . Bacteriol. 134, 200-207 (1978)l of auxotrophy resulting from the combination of two mutations, are strong supports for asparagine synthesis being an unusual biosynthetic operation. In class 11, auxotrophy results from a single mutation which leads to a modification of the efficiency of the asparaginyl-tRNA synthetase (asnRS- mutation). This auxotrophy is cancelled if asparaginase I activity (the only one present in C 1278b wild type) is cancelled by casnl- mutation. This latter mutation allows an increase in the asparagine pool which is able to compensate for the asparaginyl-tRNA synthetase partial defect of the asnRS- mutant.

Lack of information on the gene-enzyme relationship for asparagine synthesis in lower eucaryotes suggests complexities. These organisms have proved to be especially suitable for such studies [1,2].

In procaryotes an asparagine synthetase activity and mutations affecting this activity have been obtained [3,4]. The L-asparagine synthetase activity can be expressed by Eqn (1) :

NH: + ATP + L-aspartate --+ L-asparagine

-t AMP + pyrophosphate . (1)

In higher eucaryotes (plants and animal cells) an L-asparagine synthetase depending on glutamine is shown to follow Eqn (2) [5,6]:

L-glutamine + ATP + L-aspartate -+ L-asparagine

+ AMP + pyrophosphate + L-glutamate . (2)

Enzymes. L-Asparaginase I (EC 3.5.1 . l ) ; asparagine synthetase (NHi-dependent) (EC 6.3.1.1) ; asparagine synthetase (glutarnine- hydrolyzing) (EC 6.3.5.4) ; asparaginyl-tRNA synthetase (EC 6.1.1.2’2) ; aspartate transaminase or L-glutamic-oxaloacetic transaminase (EC 2.6.1.1); L-glutamate dehydrogenase (NAD’) (EC 1.4.2.2); glutaminase (EC 3.5. t.2); L-malate dehydrogenase (EC 1.1.1.37).

In this latter case NHZ can still provide the amino group of asparagine but glutamine is much more efficient.

Auxotrophs for asparagine have been observed with Neurospora crassa. They were shown to result from a single mutation which segregates as a monogenic character [7].

In Saccharomyces cerevisiae auxotrophy was obtained only recently and shown to result from the combination of two unlinked mutations asnl and asn2. Separately both mutations are not expressed in growth rate [8]. Both in Neurospora and S. cerevisiae, lack of detectable enzyme activity hampered the identifica- tion of the biochemical defect responsible for auxotrophy. Indeed the need for two mutations in the latter case indicates the existence of a complex gene- enzyme relationship which motivated the investiga- tions reported here.

In this report we show the occurrence of an asparagine synthetase in S. cerevisiae. Independently of Jones’ finding [8], we have established that total auxotrophy may result from the combination of two mutations which separately do not provoke complete auxotrophy. We show that in this case, auxotrophy is the result of a lack of asparagine synthetase. In addition

41 0 Synthesis, Activation and Auxotrophy for Aspdragine in S. cerevisiae

we show another type of auxotrophy which results from a monogenic defect affecting the asparaginyl- tRNA synthetase activity.

Preliminary results have been presented elsewhere [91.

MATERIALS AND METHODS

Reagents

L-Asparagine without free ammonia and L-glutamine with less than 2% ammonium salt were Sigma products. ~-[U-'~C)Aspartate from the Centre d'Ener- gie Atomique (France) was purified on Dowex 1, chloride form [lo]. The ~-[U-'~C]asparagine from Amersham was filtered on Whatman glass fiber paper, soaked in non-radioactive asparagine at the same concentration. L-Glutamate dehydrogenase from beef liver in 50 % glycerol, L-malate dehydrogenase from pork heart in 50 % glycerol, L-glutamate-oxaloacetate transaminase from the same tissue but in ammonium sulfate, L-asparaginase from Escherichia coli in 50 % glycerol with less than 2 % glutaminase, and yeast tRNA were Boehringer products. Nystatin was obtained from Labaz (Brussels); 1 mg contains 3000 units. Ethyl methanesulfonate was from Eastman Kodak Co.

Growth Media and Methods

The normal culture medium used in this work is designated as medium M. Medium M was prepared by mixing the following four solutions, all previously sterilized. First, medium 165, 1 1 of which contained 0.7 g MgS04.7 H20 , l g KH2P04,0.4 g CaCl2. 2H20, 0.5 g NaCl, 1 g K2S04, 10.5 g citric acid . H2O and 16 ml 10 M KOH. This medium was adjusted to pH 6.1 with further addition of KOH. It was sterilized at 121 "C for 20 min; the pH after sterilization was about 6.2. Secondly, the trace metals solution (61) was added to a final dilution of 1 ml in 1 1 of medium 165. The trace metals solution contained 10 mg H&03, 1 mg CuS04. 5 H20, 2 mg KI, 4 mg Na2Mo04.2 HzO, 14 mg ZnS04 . 7 H20, 10 g citric acid . H20, 400 mg MnS04 . H 2 0 and 5 g FeCL . 6 H20. This solution was sterilized at 121 "C for 20 min. Thirdly, vitamin solution was added to a final dilution of 10 ml in 1 1 of medium 165 containing trace metal solution 61.1 1 of vitamin solution contained 250 pg D-biotin, 100 mg thiamin . HCl, 1 g inositol, 200 mg calcium D-panto- thenate and 100 mg pyridoxine . HCl. This solution was sterilized at 112 "C for 20 min. Last, glucose was added at 3 g for 100 ml final volume by addition of 30 % sterile glucose solution to a mixture of the other three solutions.

The medium described above results from modifications of the previous procedure of this laboratory

[ll - 131. These modifications are essentially the reduction of initial concentration of metals except manganese and iron, as well as the avoidance of some unknown reaction occurring at sterilization. These modifications have been shown to reduce the lag phase of growth, which is especially important with low seeding [14].

The nitrogen nutrients absent from medium M were added separately, either (NH4)rS04 at 0.01 M or other nitrogen nutrients, usually at 1 mg x ml-l if not mentioned.

Complex medium 863 contains yeast extract, bacto- peptone and glucose [15]. Cultures were started at 2 x lo4 cells x ml-' and were collected around 3.5 x lo6 cells x ml-' (in exponential phase). Cultures were obtained at 29 f 0.5 "C on a rotary shaker. Harvesting and washing with distilled water were done in the cold and the pellets were frozen.

Cell-Free Extracts

Cell-free extracts were obtained in the cold. Cells from 500 ml of culture were supplemented with 2 ml buffer, either 0.1 M potassium phosphate pH 7.7 with 0.2 mM EDTA (MgK salt), designated as phosphate buffer, or 0.1 M Tris-HC1 buffer pH 7.5 with 0.2 mM EDTA plus 0.1 mM dithiothreitol, designated as Tris buffer. The paste was crushed with a French pressure cell and centrifuged at 27 000 x g for 20 min. The supernatant was used after gel filtration through Sephadex G-25 fine from Pharmacia in a column (6 x 1.5 cm), previously conditioned with the corresponding buffer. Protein content was about 6 - 10 mg x ml-' and was determined according to Lowry et al. [16] at a dilution such as to avoid interference from Tris and dithiothreitol. Enzyme activities were measured immediately after preparing the cell-free extract.

Enzyme Activities

The enzyme activities are always expressed in micromoles of product formed per hour per milligram of protein at 30 "C.

L-Asparaginase I

There is no asparagine I1 in the wild-type strain C1278b from this laboratory [17]. The reaction mixture contained 100 mM phosphate, 10 mM L-asparagine and 0.3 -0.4 mg protein for 1 ml of total volume. Incubation was carried out at 30 "C for times ranging between 0 and 30min and the reaction stopped by addition of 0.05 ml of 50 % trichloroacetic acid. After centrifugation, the NH; produced was estimated in 0.5 ml of supernatant, neutralized with 1 M Tris, by the glutamate dehydrogenase method [15].

.

F. Ramos and J.-M. Wiame 41 1

L-Asparagine Synthetase

This enzyme was assayed by the method previously used for animal cells [18] with slight variations. The principle is given by the following reaction :

L-Glutamine (or NH4Cl) + L- [U-14C]aspartate

+ ATP X G ~-[U-'~C]asparagine + L-glutamate

+ AMP + pyrophosphate.

Aspartate was separated from asparagine by electrophoresis in the presence of non-radioactive asparagine, detected by a slight ninhydrin test. Asparagine migrates slowly (about 1.5cm) towards the cathode and the electrophoresis paper was cut into sections of 1 cm by 3 cm. Radioactivity was measured in toluene/0.05 % PPOin aBeckmanLS-1OOC liquid scintillation counter. Aspartate moved towards the anode about 5 cm. Background values, i.e. incubation without ATP or without nitrogen donor or without Mg2+, were constant and about 0.1 % of total radioactivity present in the sample. The reaction mixture contained 100 mM phosphate buffer, 10 mM glutamine or 20 mM NH4C1, neutralized 4 mM ATP, 8 mM MgC12, neutral 4 mM [14C]aspartate (about lo3 counts x min-' x nmol-') and 0.3 mg protein for 0.1 ml total volume. Incubation at 30 "C was from 0 to 30 min, samples of 0.01 ml being streaked onto the Whatman 3MM paper in a line (3 x 20 mm) previously loaded with 0.5 nmol non-radioactive asparagine and immediately dried by air blowing. Strips (20 x 4 cm) of Whatman paper were subjected to electrophoresis in 0.04 M sodium acetate buffer (pH 5.5) for 30 min at 35 V/cm and cooled with running water.

L-Asparaginyl-tRNA Synthetase

The assay of this enzyme was similar to that described by Andrulis et al. [19] with some modifications. The incubation mixture was composed of 100 mM Tris-HC1 pH 7.5 with 0.2 mM EDTA, 40 mM KC1, 15 mM magnesium acetate, 10 mM neutral ATP, 1 mM dithiothreitol and 100 pg tRNA. L-['~C]- Asparagine (0.025-0.3 mM, 45000 counts x min-' x nmol-') and enzyme (4 - 80 pg protein, depending on whether it was obtained from wild type or mutant). The reaction was started by addition of enzyme. In- cubation lasted 2-6 min at 30 "C; 0.1-ml samples were taken and mixed with 3 ml of 10% trichloroacetic acid plus 1 mM non-radioactive asparagine at 0°C. These samples were filtered on Whatman glass fiber (GF/C) paper (2.5-cm diameter) previously washed with trichloroacetic acid plus asparagine. The filtrate was then washed five times with 5 ml cold trichloroacetic acid/asparagine mixture. Radioactivity was measured as for asparagine synthetase. The blank was obtained by addition of trichloroacetic acid at time

zero. These blanks were constant, they contained about 1/7000 of the total radioactivity of the sample and were similar to blanks obtained in reactions without ATP or without tRNA. Strains of yeast deprived of asparaginase activity eliminate the possibility of asparagine degradation.

Pool of L-Asparagine

3.5 x lo6 cells/ml culture in exponential phase were filtered on millipore filters, washed with water and quickly soaked in boiling water for 5 min. The asparagine content was estimated with asparaginase plus glutamate-oxaloacetate transaminase plus malate dehydrogenase. Aspartate present before the reaction was estimated independently with glutamate-oxaloacetate transaminase plus malate-dehydrogenase.

Strains

Unless otherwise mentioned the original wild type of this laboratory (Z1278b) and its mating type (mutant 3962c) have been used [20]. In addition to being isogenic with all of our strains, in the present case the C1278b strain was chosen for having only one asparaginase [17]. The non-isogenic strain S288C was from Fink's collection.

Mutant FR91 deprived of asparaginase I was obtained by X-ray mutagenesis [20] and replica plating of plates from solid medium M containing ammonium salt onto medium M containing L-asparagine. This type of mutant has been described [21]. The designation of this mutant as asp2 may lead to the obvious confusion with an aspartate mutant. We shall use casnl- (for catabolism of asparagine). casnl- mutation is almost certainly allelic with mutation aspl. Segregants 8521 a, 8597c and 8607d have the defect of FR91 for asparaginase I.

Auxotrophic mutants for asparagine (FR101- FR116) were obtained from two distinct mutagenic treatment with ethyl methanesulfonate [22] and se- lected after enrichment by the nystatin method [23]. After the treatment cells were grown exponentially on medium M containing L-asparagine. This population (about 3 x lo6 cells/ml) was transferred into medium M (without nitrogen) at pH 4.2 and starved for 20 h. These starved cells were transferred into medium M containing ammonium salt and L-asparagine at pH 4.2 (the usual medium is at pH 6.2) at an initial population of lo6 cells/ml. 6 pg nystatin were added when the population was 3-4 x lo6 cells/ml and after 1 h the culture was centrifuged, washed and plated onto medium M containing L-asparagine. When colonies were visible on the plate, they were replicated onto medium M contains ammonium salt and L-aspartate. The presence of aspartate eliminates aspartate-

412 Synthesis, Activation and Auxotrophy for Asparagine in S. cerevi.Tiae

Table 1, Asparagine synthetase activity in wild-type Z 12786 of S. cerevisiae Cells were grown on medium M plus glutamate. 10 pl of reaction mixture contained 27 pg protein and 36000 counts/min of L-[U'~-C]- aspartate

Reaction mixture ~

Incubation Asparagine time

Complete

- glutamine

ATP

- Mg"

min counts/min 0 36

15 330 30 615

15 31 30 43

15 31 30 34

35 30

34 45

- glutamine + NH3 15 192 30 309

transaminase-deficient mutants, pH 4.2 was used to optimize nystatin efficiency [23].

Mutants FRlOl -FR106 were obtained from one operation, mutants FR 107 - FR 1 16 from another; therefore mutants FR103 and FR107 used in this work are derived from independent mutations. Mutant FRllO is of another phenotype as described in Results.

Genetic analysis was performed as described [24].

RESULTS AND DISCUSSION

Asparagine Synthetase in S . cerevisiae

Previous attempts by Rognes to demonstrate asparagine biosynthesis in Saccharomyces cell-free extracts failed. He succeeded in demonstrating glutamine-dependent synthesis in extracts of lupin seedlings and suggested that asparaginase could interfere with detection of asparagine synthesis in vitro in yeast as already shown in E. Cali [4,6]. The usual wild-type strain of S. cerevisiae of this laboratory, C1278b, is known to possess only asparaginase I. Asparaginase 11, commonly found in membranes of most strains, is absent from C1278b [17]. Cell-free extracts of C1278b have been shown to contain asparagine synthetase activity when tested in the usual conditions, similar to those previously used for animal or plant tissues (see Methods). Asparagine synthesis is linear with time for at least 30 min. Mg2+, ATP and amide nitrogen donor are absolute requirements (Table 1). Comparison between NH; or glutamine as amide nitrogen donors shows that glutamine is more efficient. Maximal velocity is twice as good with glutamine and

/

- 1000 0 1000 2000

Fig. 1. Rec,iproculplot of L-asparagine syntheiase wtivity in bvild iype as a function of NH: (a) or L-glutamine (0) Tubstrate concentration. u is measured as Fmol L-asparagine produced x h-' x (mg protein)-

the K, for glutamine is 0.57 mM, as compared with 20 mM for NH; (see Fig. 1). Asparagine resulting from asparagine synthetase activity is not destroyed by asparaginase I in the conditions of the assay in vitro. The recovery of asparagine is not increased in asparaginase-I-less mutant (strain 8521 a, Table 2, expt 2 vs 1). However, under some experimental conditions the presence of asparaginase I may affect in vivo the availability of asparagine for protein synthesis (see below). The interference of asparaginase I1 in cell-free extracts has not been studied except for a preliminary assay. In the non-isogenic strain S288c, which is expected to have asparaginase 11, the activity is lower (6--8 times) and, as expected, this is especially so with longer incubation times as previously used in assays with other materials. The identity of the asparagine synthesis reported here with the activity needed for growth will be proved in the study of auxotrophic mutants reported in the next section.

L-Aspmagine Auxotrophs

Replica plating of an ethyl-methanesulfonate- treated wild-type suspension did not show any asparagine auxotroph. We had to use a nystatin- enrichment method (see Materials and Methods) ; 16 mutants (FR101 -FRI 16) were recovered. All grow on medium M plus asparagine and do not grow on medium M plus N H I or medium M plus NHZ + L-aspartate. One mutant, FRI 10, behaves differ- ently from the others. It does not grow on complex medium 863 [I51 supplemented with L-asparagine (1 mg x ml-') or in medium M plus glutamate and L-asparagine. A complementation test confirms that

F. Ramos and J.-M. Wiame 413

Table 2. Asparagine synthetase activity in mutant strains

Expt Strain Genotype Nitrogen nutrient in culture Specific activity Asparaginase activity

with NHZ with glutamine cf. wild type

nmol h-' (mg protein)-' x ~~~ ~~

1.

2. 3. 4. 5. 6. 7.

8.

Z 1278b

8521a 8572a 8530a 853013 8556a 8597c

8607d

wild type

casn I - asnA - - I , asnB- - I usn A - - I , + +, asnB--l

asnA--I , usnB--I, casnl- asnRS-, casnl-

US?? R S - 1

NHJ or asparagine or glutamate NHJ asparagine asparagine asparagine asparagine

glutamate + asparagine glutamate + asparagine

25 f 5 25 k 5

4 + 1 25 + 5 25 5

- < 0.2

5 0.2 25 + 5

50 10 50 k 10

5 0.2 8 + 2

50 f 10 50 k 10

5 0.5 50 + 10

100 < 2 100 100 100 100

< 2 <2

Table 3. Growth of segregants from eight tetrads issued from a cross between FR103 and wild-type 3962c Growth was measured on medium M plus NH: and L-aspartate; -, no growth; +, slow growth; +, normal growth

Tetrad type Growth Genotype Number

Table 4. Growth of segregants from 12 tetrads issued from a cross hetween 8530a and 85306 All details as in Table 3

Tetrad type Growth Genotype

of tetrad< Parental ditype k asnA-, + f asnA-, +

Parental ditype - -

+ +

asnA-, usnB- 2 asnA -, asn B +, + +. -c

+ +, asnB- + +, usnB-

Non-parental ditype - (8572a) asn A -, usn B - - (857213) asnA-,asnB- + +, +

Nan-parental i (8530a) asnA-, + 1 + +, + + (8530b) +, usnB- -

ditype + usiiA-, + Tetratype -f abnA-, + utnA- , asnB- +, urnB-, and + I { + +

+ +, a d -

& (8562a) asnA-, + +, + and

+ + I I +, nsnB-

+ Tetratype ~ (8526b) usnA-, asnB- 5

the FRllO mutant belongs to a distinct class from the others. All others belong to the same complementation class. Assays for asparagine synthetase revealed a clear distinction and allowed all mutants except FR110 to be designated as asparagine-synthetase-less mutants. FR103 and FR107 are typical cases and will be analyzed further (see next section). Mutant FR110 with normal asparagine synthetase activity will be investigated in the section after that.

L- Aspavagine-Synthetase-less Mutants

Genetic Analysis. Mutant FR103 has been crossed with wild-type 3962c. Analysis of eight tetrads based on type of growth on mediumM plus NH; and L-aspartate, reported in Table 3, exclude an auxotrophy resulting from a monogenic mutation. Partial auxotrophy occurs as a new phenotype. As expressed

in Table 3, all results could be explained on the basis of a complete auxotrophy resulting from the combination of two mutations designated as asnA- and asnB-. One of them, asnB-, gives rise to a segregant which is completely indistinguishable from the wild type ; the other, asnA -, leads to a partially auxotrophic character. Direct confirmation would result from the recovery of complete auxotrophs in segregants of a cross between a segregant (8530a) and a segregant + (8530b) of a non-parental ditype tetrad. As reported in Table4, the expected result is obtained in 8572a and 8572b segregants. This shows conclusively that two mutations are needed for complete auxotrophy. Total auxotrophs from non-parental ditypes such as 8572a and 8572b are new recombinants equivalent to the original mutant FR103. Individual mutations must be present in the parents so 8530a will be used for the study of mutation asnA- and 8530b for muta-

414 Synthesis, Activation and Auxotrophy for Asparagine in S. cerevisiue

tion asnB-. As a corollary to results discussed above, a cross between 8526a and wild-type 3962c gave only the parental ditype 2 2 : 2 + . From the same tetratype tetrad, segregant 8526b of total auxotrophy phenotype should be asnA-, asnB-, similar to initial mutant FR103. Indeed a cross of this latter mutant with the wild type results in one non-parental ditype and four tetratypes. In no case did a total auxotrophic phenotype give a simple 2 - : 2 + segregation when crossed with the wild type. Diploid asnA-, +/+, asnB- (8530a x 8530b) grows without need for L-asparagine, at a rate identical to that of the wild type on medium M plus NH:. This shows that the asnA- mutation is re- cessive. FR107 is another mutant of the same complementation class as FR103. It is also of a completely auxotrophic character, but it differs somewhat from FR 103, when segregants are compared. FR107 crossed with wild type gave four tetrads of (-, +, +, +) type when tested for growth on solid medium M plus NH;. However, quantitative measurement of growth rates in liquid medium reveals the similarity with the tetratype segregation of mutant FR103. The generation times of the four segregants of tetrad 8539 are respec- tively: (a) 2 h, (b) 2 h 42 min, (c) 2 h 03 min, (d) no growth. Segregant 8539b is not very different from wild type but is slightly bradytrophic. In the case of mutant FR103 the segregant of phenotype had a generation time of 3 h 48 min. The slow-growing segregant 8539b has been shown to have a mutation allelic with the asnA- mutation from FR103. It will be designated as asnA--2, the previous asnA- being asnA--I.

Enzymatic Analysis. L-Asparagine synthetase activity, in the simple mutant asnA--I, + or +, asnB--I, or a reconstruction of FR103, asnA--I, asnB--1, is given in Table 2 (expts 4, 5 and 3). The double mutant asnA--l, usnB--l is devoid of detectable activity (less or equal to 1 wild-type activity) with both NH: or glutamine as nitrogen donor. Alone, mutation asnA -- I (segregant 8530a), with a partial L-asparagine auxotrophy, has a clear ( 5 - 9-fold) reduction of asparagine synthetase activity, but mutant asnB--I, with no auxotrophic character, has a normal synthetase activity within our limits of precision.

A combination of asnA--1, asnB--I with mutation cusnl- leading to absence of asparaginase I activity does not modify the asparagine synthetase activity in vitro or the auxotrophy of that strain (segregant 8597c, expt 7, Table 2).

This contrasts with another type of auxotrophic mutant considered in the next section. This shows that asnA - and asnB- mutations are involved in asparagine synthetase activity. Analogy with Jones' asnl and asn2 mutations [8] is too striking to suggest other than their allelism. However, at present it is impossible to identify asnA- and asnB- as being allelic with asnl and asn2 or the reverse. This will be investigated. The

most striking result from both studies is that no single mutation is able to suppress asparagine synthetase activity. One possibility also presented by Jones is the occurrence of two asparagine synthetases. If one takes into consideration the enzyme activities reported here, and if two such enzymes are indeed present, the activity of one of these enzymes must be so low as to prevent our detecting its disappearance in the presence of the other. As reported in Table 2, this cannot be excluded. For lack of sufficient precision, we cannot be sure that enzyme activity in asnA+, usnB- has not decreased with respect to wild type by an amount approaching the residual activity found in asnA -, asnB+ strain. Other more elaborate situations may be envisioned, but the most obvious path of investiga- tion would be to see if it is possible to increase the sensitivity of detection of the synthetase corresponding to gene asnB and then to compare the properties of the two enzymes, using asnA-, usnB+ and usnAt, asnB- strains.

Auxotrophy due to a Modijkation of L-Asparaginyl-tRNA Synthetase

Physiological Aspects. Mutant FR110 crossed with wild type or with any L-asparagine-synthetase-less mutant gives prototrophic diploids. Five tetrads issued from diploids with wild-type 3962c result in regular 2 : 2 segregation when tested on solid medium M plus NH: and L-aspartate. Two segregants 8556a (mating type a) and 8559d (mating type N) contain the mutation which will be designated as asnRS--1 (for reasons developed below). The activity of L-asparagine synthetase in segregant 8556a is the same its in wild type (Table 2, expt 6 vs 1).

The need for L-asparagine of the asnRS- mutant is different from that of the double mutant asnA-, asnB-. In the presence of NH;, mutant asnRS--1 re- quires a higher concentration of asparagine than mutant FR 103 and in the presence of glutamate (0.1 %) no growth is observed even when L-asparagine is at 0.1 %. With asparagine as the only source of nitrogen the rate of growth is the same as that of wild type (Table 5) . It is known that asparagine auxotrophy may result from an excessive degradation of L-asparagine due to L-asparaginase. This was observed in animal cancer cells [25,26]. We have also mentioned that L-asparagine synthetase activity in cell-free extracts of strain S288c (which is known to have two asparaginases) is lower than in extracts of our wild- type strain C 1278 b. Indeed casnl- (asparaginase-less) mutation when combined with mutation asnRS-I re- lieves auxotrophy. It should be recalled that casnl- mu- tation did not affect the L-asparagine synthetase activity of a wild type (Table2, expt2) and did not relieve the partial auxotrophy of asnA - - I nor the complete auxotrophy of the double mutant asnA--1, asnB--l.

F. Ramos and J.-M. Wiame 415

Table 5. Rate of growth of L-asparagine auxotrophs cis a function of L-asparagine concentration NH2 concentration was 0.02 M as usual; Glu = glutamate concentration, Asn = L-asparagine concentration. The measured doubling time is a routine determination which detects only obvious variation larger than 10 min. n.g. = no growth

Nitrogen nuti lent in culture

N H i Glu Asn wild type 8572a or FR103 8556a or FRllO

Doubling time of ~~

(asnA - - I , asnB- -I) (asnRS- - I )

M w m l min ~~ _ _ ~ -

0.02 0 0 120 * 5 n g. " g 0 02 0 10 120 125 n g 0 02 0 50 120 120 n g 0 02 0 100 120 120 > 240 0 02 0 1000 120 120 144

1000 0 120 * 10 n.g. 1000 10 120 138 1000 50 120 120 1000 1000 120 120

n.g. n.g. n.g. n.g.

0 0 1000 120 120 120

Altogether these results suggest that the asnRS- mutant suffers from abnormal catabolism. This could be due to modification of asparaginase activity. How- ever, the mutation a s n R S - I is not linked to or allelic with casnl-. As shown in Fig. 2, the saturation curve of asparaginase by L-asparagine, including a slight cooperative effect [27], is not modified in mutants 8572a (asnA-, asnB-) or 8556a (asnI2Y-I). The specific activity of asparaginase I is also independent of conditions of growth [17] (Table 6). The other obvious explanation would be that the synthesis of asparaginyl- tRNA synthetase is partially impaired and needs a higher pool of asparagine to compensate for lower activity. In mutant asnRS- this could be satisfied by external asparagine supply or, as in the asnRS-, casnl- double mutant, by endogenous synthesis which would supply such a pool because of the concomittant play of a partial block in the activation enzyme and the lack of degradation of the accumulated asparagine. The study of the pool provide a first indication in favour of this explanation (Table 7). The asparagine pool in wild type growing on medium M plus NH; is too low to be detected by our method. When the mutant asnRS- begins to grow at a good rate because of asparagine supply, the pool reaches a value which is at least 20 times the normal basic level (Table 7). With the a s n R S , casnf- double mutant (8607d) this level is reached without asparagine supply, obviously because of lack of catabolism. Mutation cansl- alone (8521 a) does not provoke a detectable accumulation of asparagine in cells grown on medium M plus NH; . This suggests that asparaginase I does not waste asparagine in strain C 1278 b growing on medium M plus NH; alone, but catabolism is very active when asparagine accumulates (Table 7). Increase in generation

li ip,,,,,

OO 2 4 6 8 10 [ ~ - A s p a r a g i n e ] (mM)

Fig. 2. Sarurution of r.-cisparuginase I h j L-aspurugiiic in wild type and L-asparagine auxorroph mutants. The wild type strain C 1278b (0) and the L-asparagine auxotroph mutants 8572a (0) and 8556a (0) were grown in medium M plus L-asparagine

Table 6. L-Asparaginase I acrivity

Strain Nitrogen Asparaginase nutrient activity

pmolxh- ' x (mg protein) -'

C 1278b (wild type) NH: 2.0 glutamate 2.0 asparagine 2.3, 2.4, 2.6

8572a (asnA--I, asnB--l) asparagine 2.4

8556a (asnRS--I) asparagine 2.5. 2.5

416 Synthesis, Activation and Auxotrophy for Asparagine in S. rerevisiue

Table 7. Rate of growth as a function of the L-asparagine pool in mutants defective in asparaginase ( cam- ) and asparaginyl-tHA synthetase (asnRS - ) Rate of growth was measured as generation time; n.g. = no growth. The L-asparagine (Am) pool was measured per mg dry weight

Nitrogen nutrients Z 1278b 8521a ( c a m - ) 8556a (asnRS- - I ) 8607d (asnRS- - I , in culture casnl- )

NH$ Asn growth Asn pool growth Asn pool growth Asn pool growth Asn pool -

M P&/ml min nmol inin nmol inin nmol min nmol

0.02 0 120 O ( i 1) 120-130 O(i 1) n.g. - 138 23 0.02 10 120 8 126 56 n.g. 126 59 0.02 50 120 9 130 219 n.g. - 126 372

150 464 0.02 100 120 9 162 447 > 240 -

165 752 0.02 200 120 23 180 645 192 -

0.02 500 120 41 210 800 160 21 186 804 0.02 1000 120 47 222 920 144 25 198 790

-

-0 4 8 12 Time (min)

0 2 4 6 Time (rnin)

Fig. 3. Dependence of the L-asparaginyl-tRNA synthetase activities of strains 8521a ( A ) and 8607d ( B ) on time and amount of cell extract protein. The strains used were wild type with respect to L-asparagine synthetase. They carried a cusnl- mutation (L-asparaginase I -) in order to avoid occasional degradation of L-asparagine in vitro. This allowed comparison of the L-asparaginyl-tRNA synthetase activity of mutant 8607d (a.snRS--l) with that of the wild-type strain 8521a (USERS') in cells grown in the same medium (M + NH,f). L-Asparagine concentration in the reaction mixture was 0.1 mM

time of casnZ- and asnRS-, casnl- mutants could be due to excessive (toxic) effect of the large asparagine pool or more likely to inhibition of NH; entrance by asparagine (Table 7).

The Asparaginyl-tRNA Synthetase. The capacity to activate L-asparagine was investigated in auxo-

trophic mutant asnRS- by comparing the double mutant asnRS-, casnZ- (strain 8607d) with the control mutant casnl- (strain 8521a). In both cases, absence of asparaginase ensures the absence of secondary effects due to degradation of the substrate at high concentration. Presence of mutation casnZ- also allows growth of both strains with NH; as nitrogen nutrient. Fig. 3 shows satisfactory kinetics with respect to the amount of protein and the incubation time for the first 5 min of reaction in extract of strain 8521a and 8607d. Later the rate of aminoacylation decreases and a plateau is reached, reflecting an equilibrium between the aminnacvlation reaction and the deacylation reactions [28]. We report here the activity measured in the phase where kineticsare linearwith respect to time (2-4min). Fig. 4 expresses the apparent affinity in a reciprocal plot. The asnRS- mutation leads to an increase of K, for asparagine from 0.12 mM (in casnl- mutant) to 1.1 mM in the asnRS-, casnl- double mutant. From the same figure, the extrapolated value of V of the mutated L-asparaginyl-tRNA synthetase is about three times lower than that of the normal enzyme. These results leave no doubt that the origin of the auxotrophy is a mutation in the gene coding for L-asparaginyl-tRNA synthetase.

CONCLUSIONS

L-Asparagine synthetase of the glutamine-dependent type has been shown to be present in yeast. This suggests a great homogeneity in the type of asparagine synthetase in eucaryotic cells.

Mutants described by Jones [8] and in this paper have established a first step in the gene-enzyme relationship and together stress an unusual situation. As shown here, mutations which lead to total disappearance of the a formentioned activity are double mutants ( a s n K , asnA -). Jones' double mutants tested by total auxotrophy are probably similar. The oc-

F. Ramos and J.-M. Wiame

-10 0 10 20 30 40 [ ~ - A s p a r a g i n e ] - ’ (rnM-’)

[ L - A s para g i n e ] -’ ( rnM

Fig. 4. Reciprocal plot of L-asparaginyl-tRNA .syntlieiase activitj, as ufunction of kasparagine concentration. (A) Strain 8521a ( c a m - ) ; (B) strain 8607d (casnl-, amRS-- I ) . L‘ is measured as pmol L-asparaginyl-tRNA produced x h-’ x (mg protein)-’

currence of a case of monogenic asparagine auxotrophy presented here is shown to be due to a defect in L-asparaginyl-tRNA synthetase (asnRS- mutant).

Animal cells and bacterial mutants contract total auxotrophy by a single mutation [4,29]. Monogenic auxotrophy in Neurnspora [7] could be due to a defect in the activation of asparagine as reported here. Auxotrophy which originates because of an asparaginyl-tRNA synthetase defect and its suppression by L-asparaginase I mutation illustrates as typical case of anabolism and catabolism interference.

We thank E. Dubois and C. Hennaut for discussion and K. Bro- man for reading the manuscript. This work was supported by contract 2.4542175 with the Fonds de la Recherche Fondurnentale Collective.

417

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Synthesis and Activation of Asparagine in Asparagine Auxotrophs of Saccharomyces cerevisiae