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Eur. J. Biochem. 108, 373-377 (1980) 0 by FEBS 1980 Two Asparagine Synthetases in Succhuromyces cerevisiue Fernando RAMOS and Jean-Marie WIAME Laboratoire de Microbiologie, Facult6 des Sciences, Universite Libre de Bruxelles, and Institut de Recherches du Centre d’Enseigncment et de Recherches des Industries Alimenlaires et Chimiques, Bruxelles (Received February 25, 1980) In Saccharomyces cerevisiae L-asparagine auxotrophy, resulting from a lack of L-asparagine synthesis, needs simultaneous defects in two genes. This unusual situation is shown to result from the occurrence of two L-asparagine synthetases. The meaning of this duplication remains obscure. The properties of the two enzymes are remarkably similar: both have a molecular weight of 150000 and they exhibit a slight repression and a similar inhibition by asparagine. Neither one is located in mitochondria and both are probably cytosolic. Their identification so far lies only in their behaviour on ion-exchange chromatography and in the encoding genes. Very few reports of asparagine auxotrophs mutants in fungi have appeared in the past [1,2]. In addition, attempts to disclose an enzyme ac- tivity responsible for asparagine synthesis in these organisms has failed [3]. This situation recently promoted an attempt to find the nature of the bio- chemical defect which could arise in new auxotrophic mutants. It has been shown that auxotrophy may result from two different biochemical defects [4]. First, a partial defect in asparaginyl-tRNA syn- thetase leads to obligate needs of L-asparagine for growth in minimal (ammonium) medium. This need is cancelled in strains with an additional mutation which abolishes the asparaginase activity (casnl-). The asparaginyl-tRNA synthetase mutation (asnRS-) segregates as a monogenic character [4]. This be- haviour strikingly illustrates the dramatic effect which may arise from conditions leading to a futile cycle. Second, auxotrophy may result from the cumula- tion of two recessive and genetically unlinked asnA and asnB- mutations [4], very likely allelic with asnl and asn2 mutations reported by Jones [5]. The double mutant asnA -, asnB- is totally deprived of asparagine synthetase activity using preferentially glutamine amide nitrogen as a donor of asparagine amide (eucaryotic type). A partial auxotrophy and a reduced Enzymes. Acetylglutamyl-phosphate reductase or N-acetyl-L- glutamate-5-semialdehyde: NADP oxidoreductase (phosphorylat- ing) (EC 1.2.1.38); alkaline pbosphatase (EC 3.1.3.1); L-aspara- ginase I (EC 3.5.1.1);asparagine synthetase (glutamine-hydrolyzing) (EC 6.3.5.4); asparaginyl-tRNA synthetase (EC 6.1.1.22); catalase (EC 1.11.1.6); ornithine carbamoyltransferase (EC 2.1.3.3). synthetase activity is observed in the nsnA - mutant. The case of the asnB- mutant was not clear and did not explain the meaning of the two mutations. In addition neither Jones’ asnl nor the asn2 mutation led to detectable auxotrophy [5]. Here we show that the peculiarity of L-asparagine biosynthesis in Sacclzaromyces cerevisiue is due to the occurrence of two synthetases. MATERIALS AND METHODS Reagents DEAE-Sephadex A-25 and Sephadex G-200 super- fine were obtained from Pharmacia (Uppsala, Swe- den); for other reagents, see [4]. Struins und Cultures Strains and cultures were as described in [4]. Assay of L-uspurugine Syntlietase Enzyme assay was as described in [4]. Ion-Exchange Cliromatograplzy All operations are performed at about 4°C. 20 g fresh weight cells from 10 1 exponentially growing culture in minimal (NHZ) medium [4] are suspended in 20 ml 100 mM potassium phosphate buffer, pH 7.7, 1 mM EDTA (MgK salt), 2 mM dithiothreitol. After crushing in a French pressure cell the debris and lipids

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Page 1: Two Asparagine Synthetases in Saccharomyces cerevisiae

Eur. J. Biochem. 108, 373-377 (1980) 0 by FEBS 1980

Two Asparagine Synthetases in Succhuromyces cerevisiue

Fernando RAMOS and Jean-Marie WIAME

Laboratoire de Microbiologie, Facult6 des Sciences, Universite Libre de Bruxelles, and Institut de Recherches du Centre d’Enseigncment et de Recherches des Industries Alimenlaires et Chimiques, Bruxelles

(Received February 25, 1980)

In Saccharomyces cerevisiae L-asparagine auxotrophy, resulting from a lack of L-asparagine synthesis, needs simultaneous defects in two genes. This unusual situation is shown to result from the occurrence of two L-asparagine synthetases.

The meaning of this duplication remains obscure. The properties of the two enzymes are remarkably similar: both have a molecular weight of 150000 and they exhibit a slight repression and a similar inhibition by asparagine. Neither one is located in mitochondria and both are probably cytosolic. Their identification so far lies only in their behaviour on ion-exchange chromatography and in the encoding genes.

Very few reports of asparagine auxotrophs mutants in fungi have appeared in the past [1,2].

In addition, attempts to disclose an enzyme ac- tivity responsible for asparagine synthesis in these organisms has failed [3]. This situation recently promoted an attempt to find the nature of the bio- chemical defect which could arise in new auxotrophic mutants. It has been shown that auxotrophy may result from two different biochemical defects [4].

First, a partial defect in asparaginyl-tRNA syn- thetase leads to obligate needs of L-asparagine for growth in minimal (ammonium) medium. This need is cancelled in strains with an additional mutation which abolishes the asparaginase activity (casnl-). The asparaginyl-tRNA synthetase mutation (asnRS-) segregates as a monogenic character [4]. This be- haviour strikingly illustrates the dramatic effect which may arise from conditions leading to a futile cycle.

Second, auxotrophy may result from the cumula- tion of two recessive and genetically unlinked asnA and asnB- mutations [4], very likely allelic with asnl and asn2 mutations reported by Jones [5]. The double mutant asnA -, asnB- is totally deprived of asparagine synthetase activity using preferentially glutamine amide nitrogen as a donor of asparagine amide (eucaryotic type). A partial auxotrophy and a reduced

Enzymes. Acetylglutamyl-phosphate reductase or N-acetyl-L- glutamate-5-semialdehyde: NADP + oxidoreductase (phosphorylat- ing) (EC 1.2.1.38); alkaline pbosphatase (EC 3.1.3.1); L-aspara- ginase I (EC 3.5.1.1); asparagine synthetase (glutamine-hydrolyzing) (EC 6.3.5.4); asparaginyl-tRNA synthetase (EC 6.1.1.22); catalase (EC 1.11.1.6); ornithine carbamoyltransferase (EC 2.1.3.3).

synthetase activity is observed in the nsnA - mutant. The case of the asnB- mutant was not clear and did not explain the meaning of the two mutations. In addition neither Jones’ asnl nor the asn2 mutation led to detectable auxotrophy [5].

Here we show that the peculiarity of L-asparagine biosynthesis in Sacclzaromyces cerevisiue is due to the occurrence of two synthetases.

MATERIALS AND METHODS

Reagents

DEAE-Sephadex A-25 and Sephadex G-200 super- fine were obtained from Pharmacia (Uppsala, Swe- den); for other reagents, see [4].

Struins und Cultures

Strains and cultures were as described in [4].

Assay of L-uspurugine Syntlietase

Enzyme assay was as described in [4].

Ion- Exchange Cliromatograplzy

All operations are performed at about 4°C. 20 g fresh weight cells from 10 1 exponentially growing culture in minimal (NHZ) medium [4] are suspended in 20 ml 100 mM potassium phosphate buffer, pH 7.7, 1 mM EDTA (MgK salt), 2 mM dithiothreitol. After crushing in a French pressure cell the debris and lipids

Page 2: Two Asparagine Synthetases in Saccharomyces cerevisiae

374 Two Asparagine Synthetases in Sacckarnmpres cerevisiae

are removed by centrifugation at 30000 x g for 20 min. Nucleic acids are precipitated by addition of 0.2 g neutralized protamine sulfate, stirred for 30 min, and centrifuged.

The ammonium sulfate precipitate between 40 (x and 60% saturation, is redissolved in 2 ml 250 mM potassium phosphate, 0.5 mM EDTA, 1 mM dithio- threitol and dialyzed against 20 mM potassium phos- phate with protectors. About 150- 200 mg proteins are present at this stage in 3 ml, the specific activity of asparagine synthetase is about 3 times that of the crude extract. The total sample, 150-200 mg proteins, is layered on a DEAE-Sephadex A-25 column (25 x 1.6 cm). The void volume is 24 ml. The gel is equil- ibrated with 20 mM potassium phosphate buffer, pH 7.7, containing the protectors mentioned above. Elution is obtained by a linear gradient of 50 - 200 mM potassium phosphate, pH 7.7, with protectors. Frac- tions of 4 in1 are collected and 0.02-ml samples plus 0.03 ml reaction mixture are incubated at 30°C for 30 min, The reaction is ended by 5 pl 15 % trichloro- acetic acid. 10-1-1.1 samples are then used for electro- phoretic separation of [14C]asparagine from ['4C]as- partate (3500 counts min-l nniol-I).

Molecular Weight by Gel Filtration

Samples are prepared as described for ion-ex- change chromatography (about 100 mg proteins for strain 8521a and 8539c, and 200 mg for 8539b).

The samples, dissolved in 1 ml potassium phos- phate buffer 50 mM, pH 7.7 with protectors, are layered on a Sephadex G-200 superfine column (30 x 2.6 cm). The void volume is 50 nil. Elution by the same buffered solution proceeds at a rate of 0.07 ml/ min and 1.5-ml fractions are collected. Molecular weight markers are : bovine liver catalase (240000) and Eschericl~ia coli alkaline phosphatase (78 000) from Worthington Biochemical Corporation (Free- hold, NJ, U.S.A.); Pseudornonas putida catabolic ornithine carbamoyltransferase (420000) from this laboratory (B. Wargnies) and Sacclzarom,yces cer- evisiae ornithine carbamoyltransferase (125 000), a normal enzyme from the sample. These enzymes are assayed following [6].

RESULTS

Evidence,fov Tic0 Enzymes of Similur Size

The DEAE-Sephadex A-25 chromatography re- solved the asparagine synthetase from wild-type sample in two distinct peaks of activity (Fig. 1 a). The mutant m n A - presents only the first peak of activity (Fig. 1 b) while in mutant asnB-, the second peak alone is present (Fig. 1 c). This strongly suggests an asparagine synthetase B present in asnA- (ab-

16 32 4a 64 Fraction number

2003

x 5 ._ m c

m

0 16 32 48 64

Fraction number

, , I

16 32 48 64 Fraction number

Fig. 1. Distribution of ci.piruguir ,sjwthrtci.se.r A and B activities in a DEAE-Sephudex A-25 c l~romatograp l~~~ q f a partially purified extract qf Saccharomyces cerevisiae. (a) Strain 8521a (usnA+, cisnE+, c a m - ) ; (b) strain 8539b (asnA--.?, usnB'); (c) strain 84.39~ (u.snA+, usnB--2)

Page 3: Two Asparagine Synthetases in Saccharomyces cerevisiae

F. Ramos and J.-M. Wiame

100 - 6 80 ._ m 3

37s

- -

15

10

I I I I 1.4 1.6 1.8

41 vo Fig. 2. Gel .fi'ltration on calibrured Seplzadex G-200 column, of u partially purified extract oJ' Saccharomyces cerevisiae. The arrow indicates the apparent iuolecular weights of asparagine synthetases in wild type (strain 8521a) and in the two mutants (8539b and 8539~). V,, elution volume; V,, exclusion volume

breviated A-) mutant, and an asparagine synthetase A present in asnB- (abbreviated B-) mutant. There is no indication of hybrid molecules.

The size of the two synthetases has been appre- ciated by molecular sieving (see Materials and Meth- ods). The apparent molecular weights in wild-type and in the two mutants are indistinguishable within the limit of experimental errors: 150000f5000 in A'B', A'B- and B'A- strains (Fig.2).

Regulation

In the absence of regulation of enzyme synthesis and if there is no secondary effect due to individual mutation, the activity in the wild type should be the sum of the activities in mutants A-B' and A+B- . As shown in Table 1, this is true when cells are grown on asparagine. A-B' + A'B- activity is 64 units, in the wild type (A'B') it is 63 units. However, this does not remain true when cells are grown on minimal medium (NHZ), the sum of activities in mutants is significantly higher (80 units) than in the wild type (55 units). When modifications of the activities in the two culture media are compared it is clear that the higher activity with NH4' is due to A-B', in which growth on asparagine reduces the activity from 34 units to 15. A'B- mutant activity remains the same in both conditions of culture. A'B- has a higher activity (about 49 units) and does not show a growth rate limitation; A-B+ is slightly auxotrophic (limited in asparagine synthesis) [4]. In this last condition

Table 1. i.-Aspuragine synthetase activity in asnA- and asnB- mutants NHZ is 20 mM and asparagine 1 mg/ml

~~~~

Strains and genome Nitrogen Specific activity of nutrients asparagine synthetase

2'1278b (wild type) 8539b (A ~ -2, B') 8539c (A', B--2) Diploids 8539cx8539d (A', B--2/A--2, B--2) 8539cx8630a (A', B--2/A+, B--2)

N H4' asparagine NHZ asparagine NHZ asparagine

NHf asparagine NHZ asparagine

nmol h- ' mg protein-'

5 5 k 5 63+6 34+4 1 5 k 3 46 49

41 26 50 53

Table 2. Inliibition of the asparagine syntlzetases by asparagine Asparagine casnl- mutation is included to avoid any degradation of added asparagine

Aspar- Sirain 862Yh ( A + B - - 2 , Strain 8629a ( A - - 2 , B + agine c a d - ) casnl-)

~ ~ -~ ~

activity inhibition activity inhibition

mM counts/min "/, counts/min 'i: 0 1540 0 930 0 0.4 1250 19 860 8 4 280 82 290 69

20 70 96 80 91

one may explain the highest activity on NH4f by a release of end-product repression (from 15 to 34 units). As the wild type is not repressible by aspara- gine, one may expect that endogenous asparagine synthesis already produces a state of full repression.

Absence of repression in A'B- mutant could arise because asparagine synthetase is not repressible or because endogenous asparagine synthesis in that case is also sufficient for repression.

To choose between the two possibilities, a diploid with half the A + gene (A'B-/A-B-) has been com- pared to the A+B-/A'B- diploid. As expected, the latter has an activity identical with haploid A + B - but the former has more than half the activity when grown on N H:, showing derepression due to limita- tion of asparagine synthesis and a slight but highly significant increase of the generation time of 10 + 2 min. As expected, when grown on asparagine A+B-/A-B- is half as active as A'B-/A'B- (Table 1).

Although weak, the regulation of both enzymes is obviously similar and dose not disclose any physio-

Page 4: Two Asparagine Synthetases in Saccharomyces cerevisiae

3 76 Two Asparagine Synthetases in Saccharomyces cerevisiae

Table 3. Suhcellular localization of' the asparagine syntiietuses Fractions I, 11, and IIb as defined by Jauniaux et al. [lo]

Source Fractions Proteins Volume Enzyme activity

asparagine synthetase acetylglutamate ornithine carbamoyl- phosphate reductase transferase

total specific total specific total specific activity activity activity activity activity activity ( x 10-3) ( x 10-3)

ml pmol mg protein-' h- ' ~~ ~~~~~ ~~ .~

mg

From strain I crude extract 246 30 7134 29 541 2.1 3690 15 8539b (A--2, 11, soluble 228 30 7980 39 228 1 .0 3420 1s B') grown IIb mitochondria1 39 5 190 5 418 11 114 3 with NH;

From strain I crude extract 192 30 7104 37 8539d (A', 11, soluble 168 30 7056 42

with NH; B--2) grown IIb mitochondrial 12 10 60 5

logical meaning for this enzyme duplication. Absence of an effect of Jones' asnl and asn2 mutation on the growth rate may be due to a higher asparagine synthe- tase activity in this strain, which is not isogenic with our wild type (C 1278 b).

That full repression of the biosynthetic enzyme is achieved by the endogenous production of the end- product is not uncommon and was observed in the classical study of the regulation of the Salmonella typhimurium histidine operon [7], in contrast to arginine regulation in Eschericlzia coli [8] and in yeast [9].

To pursue the distinction between both enzymes the possibility of end-product inhibition has been measured. As reported in Table 2, both enzymes are similarly inhibited by asparagine ; 50 inhibition occurs at 1 mM. This is above the maximal apprecia- tion of the endogenous pool in the wild type and this inhibition is not likely to have a regulatory function. However, it may explain the fact that the first class of mutant (asnRS-), with a partial defect in asparagine activation, does not recover a normal rate of growth on minimal medium when the pool of asparagine is allowed to increase at least 20 times, because aspara- ginase activity is cancelled by the cusnl- mutation [4].

The Subcellular Localization of the Asparagine Synthetases

The localization of the enzymes was investigated following the method of Jauniaux et al. [lo]. Results are reported in Table 3 . Acetylglutamate-phosphate reductase is used as a mitochondrial marker [lo] and ornithine carbamoyltransferase as a cytosolic marker

CONCLUSION AND DISCUSSION

Saccharomyces cerevisiue has two distinct aspara- gine synthtases of the eucaryotic type (glutamine- dependent) encoded by two unlinked genes.

In yeast, so fas as we know, asparagine as a specific element is only used for protein synthesis. In addition, unlike some plants [12], yeast does not accumulate endogenous asparagine ; this amino acid concentra- tion is indeed one of the lowest.

Enzyme multiplication is a common situation in branched pathways [13,13a]. Yet, to our knowledge the duplication of the final enzyme of a biosynthetic pathway has only been observed in the case of S-adenosylmethionine synthesis in S. cerevisiue [14]. In this last case one may foresee distinct functions. A search for a physiological distinction between the two asparagine synthetases, including regulation of synthesis, regulation of activity and subcellular lo- calization, as well as the size of the enzymes, indicates a striking similarity between these two enzymes, and the meaning of this duplication remains obscure. Duplication of ornithine carbamoyltransferase in some E. coli, midway in the arginine biosynthesis, also remains an open question [15].

We thank C. Legrain and J.-C. Jauniaux for advice. This work was supported by the Fonds de la Recherche Fondumentulr Collective, grant 2.4529.79.

REFERENCES

1. Beadle, G. W. & Tatum, E. L. (1945) Am. 1. Bot. 32,678-686. 2. Arst, H. N., Kinghorn, J. R. & Drainas, C. (1977) in Genetics

andPliysiology of Aspergillus (Smith, J. E. & Patemand, J. A,, eds) p. 156, Academic Press, London and New York.

Page 5: Two Asparagine Synthetases in Saccharomyces cerevisiae

F. Ramos and J.-M. Wiame 371

3. Rognes, S. E. (1970) FEBS Lett. 10, 62-66. 4. Ramos, F. & Wiame, J.-M. (1979) Eur. J . Bioclzem. 94, 409-

5. Jones, G. E. (1978) J . Bacteriol. 134, 200-207. 6. Legrain, C., Stalon, V., Noullez, J. P., Mercenier, A, , Simon,

J . P., Broman, K. & Wiame, J.-M. (1977) Eur. J . Biockem. 80,

7. Ames, B. N. & G a r y , B. J. (1959) Pror. Natl Acad. Sci. U.S .A .

8. Gorini, L. & Maas, W. K . (1957) Biockim. Biophys. Acta, 25,

9. Bechet, J., Grenson, M. & Wiame, J.-M. (1970) Eur. J . Biochem.

10. Jauniaux, J . C., Urrestarazu, L. A. & Wiame, J.-M. (1978)

41 7.

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45, 1453-1461.

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J . Bucteriol. 133, 1096 - 11 07.

11. Urrestarazu, L. A,, Vissers, S. & Wiame, J.-M. (1977) Eur. J . Biocliem. 79, 473-481.

12. Scott, B. D., Farnden, K. J. F. & Robertson, J. G. (1976) Nu- ture (Lond.) 263, 703 - 705.

13. Stadtman, E. R., Cohen, G. N., Le Bras, G. & Robichon-Szul- majster, H. (1961) ColdSpring Harbor Sq’mp. Quunt. Bid. 26,

13a. Stadtman, E. R. (1970) in The Enzymes (Boyer, P. D., ed.) vol. 1, pp. 397-459, Academic Press, New York and Lon- don.

14. Cherest, H., Surdin-Kerjean, Y. Exinger, F. & Lacroute, F. (1978) Mol. Gen. Genet. 163, 153-167.

15. Legrain, C., Stalon, V. & Glansdorff, N. (1976) J . Bucteriol.

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F. Ramos, Laboratoire de Microbiologie, Faculte des Sciences de I’Universittt Libre dc Bruxelles, Avenue Emile-Gryzon 1, 8-1070 Bruxelles, Belgium

J.-M. Wiame, Institut de Recherches du Centre d’Enseignement et de Recherches des Industries Alimentaires et Chimiques, Avenue Emile-Gryzon 1, B-1070 Bruxelles, Belgium

Note Added in Proof. The existence of two separate genes coding for asparagine synthetases in E. cali has been reported very recently [Felton, J., Michaelis, S. and Wright, A. (1980) J . Bacteriol. 142, 221 -2281. Although a direct characterization of the two pro- teins is not shown, this is likely to bc due to enzyme instability [Humbert, R . and Simoni, R . D. (1980)J. Bucteriol. 142,212-2201, One may conclude that duplication of genes coding for asparagine synthetases is very general and not limited to fungi