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Eur. J. Biochem. 48, 603-616 (1974)

The Participation of the Anabolic Glutamate Dehydrogenase in the Nitrogen Catabolite Repression of Arginase in Saccharomyces cerevisiae

Evelyne DUBOIS, Marcelle GRENSON, and Jean-Marie WIAME

Laboratoire de Microbiologie de l’universite Libre de Bruxelles, and Institut de Recherches du Centre d’Enseignement et le Recherches des Industries Alimentaires et Chimiques, Bruxelles

(Received April 5 /July 8, 1974)

Arginase synthesis appears to be under the control of at least two distinct processes. In addition to substrate induction which operates through a Jacob and Monod mechanism, one may define a process of nitrogen catabolite repression which is a part of the ammonium effect. The two processes show a large degree of independence most specifically shown by mutations which independently cancel each of the two mechanisms without affecting the gross physiology. In this line we show that controversial conclusions can be reached when using different approaches to provoke catabolite derepression.

The nitrogen catabolite repression is abolished by the gdhA - mutation which affects the structural gene for the NADP-specific glutamate dehydrogenase.

The effect of the gdhA- mutation is retained when the metabolic defect is compensated by addition of glutamate as well as the best nitrogen nutrients, glutamine and asparagine.

This control of arginase and of some, but not all, other nitrogen catabolic enzymes may operate at constant levels of glutamate dehydrogenase (NADP). It occurs when the glutamate dehydrogenase (NADP) is in its catalytically active state, which requires the simultaneous presence of two substrates, NH: and 2-oxoglutarate. In fungi the glutamate dehydrogenase (NADP) is the first enzyme of the assimilatory pathway of ammonium, which, when functioning, makes arginase unnecessary. However the regulatory function seems to operate by an intrinsic modification of the enzyme itself rather than by the result of its catalytic action.

Carbon catabolite repression appears largely independent from nitrogen catabolite repression and vice versa. However, arginase synthesis is enhanced in mutants lacking aconitase as well as by very poor carbon nutrition. Both conditions decrease the level of 2-oxoglutarate which is required for the expression of nitrogen catabolite repression.

Once specific induction and nitrogen catabolite repression are clearly defined, it becomes possible to show that additional regulatory processes are involved in arginase synthesis (Wiame, 1973).

Enzyrncx Aconitase or citrate (isocitrate) hydro-lyase (EC 4.2.1.3); allantoinase or allantoin amidohydrolase (EC 3.5.2.5); arginase or L-arginine amidino-hydrolase (EC 3.5.3.1): cx-glucosidase or a,D-glucoside glucohydrolase (EC 3.2.1.20); glutamate dehydrogenase (NADP’) or L-glutamate : NADP’ oxidoreductase (deaminating (EC 1.4.1.4) ; glutamate dehydrogenase (NAD’) or L-glutamate : NAD +

oxidoreductase (deaminating) (EC 1.4.1.2) ; glutamate syn-

thase or L-glutamine : 2-oxoglutardte aminotransferdse (NADPH-oxidizing) (EC 2.6.1.53); glutamine synthetase or L-glutamate :ammonia ligase (EC 6.3.1.2); histidase or L-histidine ammonia-lyase (EC 4.3.1.3) : ornithine carbamoyltrans- ferase or carbamoylphosphate : L-ornithine cdrbamoykrans- ferase (EC 2.1.3.3); Spornithine transaminase or ornithine-2 oxoacid aminotransferase (EC 2.6.1.13): urea amidolyase or urea : carbon-dioxide ligase (ADN-forming) (decarboxylating, deaminating) (EC 6.3.4.6).

Eur. J. Biochem. 48 (1974)

604 Nitrogen Catabolite Repression of Arginase in S. cerevisiue

The availability of a nutrient and the need for it are the two logical conditions which have been shown to control the function of the catabolic pathway of that nutrient. “Diauxies” illustrate the existence of this double control [2]. Usually both physiological conditions are required for the full promotion of the synthesis of an enzyme which belongs to such a pathway [3,4]. This applies to the catabolism of nitrogen compounds as well as to the supply of carbon (and energy) nutrients [4,5].

For the lactose metabolism in Escherichia coli, it is clear that two distinct mechanisms are involved in the two physiological functions. The operator gene provides a lock which is closed by negative control and opened by the availability of a nutrient or one of their derivatives (induction) [6]. The promoter gene is the site of a positive control which signals the need by being produced in the absence of a “good nutrient” such as glucose [7- 101. Practically, it is important to recognize that even simple modifications of physiological conditions can result in the triggering of more than one mechanism [ I l l and a genetical approach can help to distinguish the parts which contribute to an overall process.

Although some nitrogen catabolic enzymes involved in the metabolism of glutamate in bacteria appear to be under the control of adenosine 3‘: 5‘- monophosphate (cyclic AMP) [12], it is clear that another factor is involved in the control of the synthesis of histidase by ammonia [13].

Mutations which affect the utilization of arginine as nitrogen nutrient in yeast open the possibility of analyzing the parts of the overall process which governs the synthesis of arginine catabolic enzymes. In this paper we shall be concerned with arginase, the first enzyme of the pathway.

In S. cerevisiae mutations indicate the existence of operator genes for a biosynthetic[14] as well as for catabolic en7ymes [15]. The two first enzymes of the catabolism of arginine, arginase and G,L-ornithine transaminase, are coded by two unlinked genes cargA and cargB, and the respective operators are defined by the two operator constitutive mutations cargA+ 0- and cargB+ 0- . Mutation cargA’ 0- confers high level constitutivity specifically to arginase with the useful property that the level of that enzyme is not modified by addition of arginine or other inducers to minimal (NH:) medium.

Other mutations designated by argR- [16] pleio- tropically keep arginase and transaminase syntheses at their lowest level. For arginase the level is half of the one observed in non-induced wild-type. As added inducer does not modify arginase synthesis, one may say that induction either by endogeneous or added inducer has been lost. These mutations are not of the

is type of the lactose system in E. coli [6]. They suggest the existence of a common element: an “ambivalent” repressor which operates on both the anabolic and the catabolic concurrent pathways. This ambivalent repressor appears to be the common entrance for the signal of repression and induction by arginine, ornithine and their analogues, and in the case of induction this activated ambivalent repressor acts itself as an inducer.

Induction appears as a cascade process in two steps [15] (Fig. 1). Whatever being the mechanism, for our present concern, such process offers the possibility of freezing the production of arginase at two extreme levels : the lowest level by mutation of argR and a high level by mutation of the operator of arginase. Muta- tions affecting the specific repressor of catabolic enzymes are not as useful because they retain partial inducibility.

A third type of mutation (gdhA-) located in the structural gene for the NADP-anabolic glutamate dehydrogenase suppresses the inhibition of NH fq on a number of catabolic functions, such as the activity of the general amino acid permease and the synthesis of arginase, allantoinase and urea amidolyase. A most important fact is that the supply of glutamate in addition to NH fq, which compensates for the catalytic defect produced by the mutation, does not restore repression of these enzymes. At least one other catabolic enzyme, the NAD-specific glutamate dehydrogenase, does not loose its NH; control as a result of thegdhhA- mutation [17- 191.

From these works it was concluded that the anabolic glutamate dehydrogenase (NADP) does not regulate NH: repression by its catalytic capacity to form glutamate and it was proposed, among different possibilities, that the enzyme itself with proper effector(s) may be a regulator [17- 191.

The release of NH; effect on the synthesis of catabolic enzymes by the same type of mutation has been confirmed in Aspergillus nidulans by Arst and McDonald [20] and others [21]. Recently Hynes has shown that glutamate dehydrogenase (NADP) activity is lost by carbon starvation in A . nidulans. This explains how enzymes of nitrogen catabolic pathways may be affected secondarily by carbon nutrition [22]. Glutamate dehydrogenase (NADP) is also inactivated in A . nidulans when glutamate replaces glucose as carbon source [23] and this allows interpretation of the permeability changes observed in the same way as in the gdhA- mutants of Saccharomvces cerevisiae [ 171.

The purpose of this paper is to define a nitrogen catabolite repression of arginase and to analyze a part of the regulatory mechanism exerted by the glutamate dehydrogenase (NADP) in that process.


E. Dubois, M. Grenson, and J.-M. Wiame 605

Arginine Towards genes of anabolic pathway ornithine

efc. / ARGR -L ARGR* Inactive Active

ambivalent repressor ambivalent repressor \i CAR\* : AcYv;: repressor of catabolic

O i CA RGR Inactive repressor of catabolic enzymes Arginase

Fig. 1. Cascade control of the arginine catabolic enzymes of S. cerevisiae. Entrance of the metabolic signals into regulatory circuits is common for induction of catabolism and repression of anabolism. When activated, the common element (ARGR) designated as ambivalent repressor, plays the role of inhibitor (or repressor) of the repressor CA RGR specific for the catabolic pathway

MATERIALS AND METHODS

Cultures

Cultures are grown under conditions described before [14] in medium no. 150 containing salts, buffer- ed at pH 6.1 with citrate. Medium no. 150 supple- mented with glucose and vitamins and no source of nitrogen will be referred to as medium M.

The nitrogen compounds added are specified. NH: is supplied as 0.02 M (NH4)2S04, and amino acids as 1 mg/ml of the L isomer. When these con- centrations are altered, the amount is given in brackets, in pg/ml. Changes in carbon source are mentioned explicitly. Medium no. 863 contains 1 % yeast extract (Difco), 1 % bactopeptone. 2% glucose is added after sterilization. Cells are harvested in exponential phase of growth and not higher than 0.6 mg cells dry weight per ml. An unbalanced protein and enzyme synthesis can occur at higher populations densities, even with an apparent late exponential phase as judged by corrected absorbance measurements.

For growth in chemostat, a growth vessel described in Fig.2 is used. The concentration of (NH4)2S04 is reduced to 0.5 mM. The content of the growth vessel, about 200 ml, is collected after the stabilization of the turbidity, usually after 2 days. 2 pg cycloheximide per ml are added before harvesting.

Strains

Most strains derive from the same wild type C12780b (a) and its mating-type mutant 3962c (a); they are thus in principle isogenic except for the mutations mentioned. However, to avoid double

Per 1st ai t ic PUT,?

from a 3 l i ters fresh medium stock

t to sink or collector

Fig.2. Culture vessel for chemostat. The fresh medium con- tainer and the culture vessel, connected by tubing are sterilized together

mutants, reconstructed segregants are preferred to original mutants.

Mutant BJ210 bears the argRZZ--10 mutation; it is an argR- mutant of class I1 (arabic numbers differentiate individual mutation of a given class) [16]. Mutation argAf 0--2 present in segregant 7204b is one of the constitutive operator mutations for arginase [ 151.

gdhA--1 mutation present in segregant 4324c is a mutation which abolishes NADP-specific glutamate dehydrogenase activity [17,19]. The mutant MG1748 has an aconitase activity reduced to 20% of the wild type. It has been selected by its slow growth on NH; as nitrogen nutrient; normal growth is recovered by addition of glutamate. Strain AG2 is a mutant lacking arginase [24]. Strain FLlOO is the usual wild type Lacroute's laboratory [25], S288c was obtained from G.Fink [26] and H 1326 was provided by R. Hutter (Eidgenossische Technische Hochschule, Zurich). Tetraploids are constructed following the method described by F. Hilger [27].

ED220, ED300, ED301, ED302, ED303, ED304 and ED309 are revertants of gdhA-. For the strains MG1749, 51414d, 50821a, see [19].


606 Nitrogen Catabolite Repression of Arginase in S . cerevisiue

Enzyme Assays

Arginase, glutamate dehydrogenase, aconitase and a-glucosidase activities were measured following [28, 29,181 respectively, and rate of uptake as in [30].

Pool of Metabolites

Arginine is estimated by Sakaguchi reaction [31]. The pool of NH; and 2-oxoglutarate are extracted from cold washed cells (about 200 mg dry weight) resuspended in 8 ml of 0.3 M HC104 for 1 h at 0°C and afterwards neutralized to pH 7.6 with about 2 ml 1 mM K3P04. The potassium perchlorate precipitate is eliminated together with the cells by centrifugation. 2-Oxoglutarate is measured enzymatically with com- mercial beef glutamate dehydrogenase following the oxidation of NADP spectrophotometrically [32]. NH '4 is measured by the same method or with an NHf, electrode (Orion Research Inc.)[33]. The NADP and NADPH are extracted from cells collected on Milli- pore filter and quickly washed with cold water. NADPH is extracted under conditions which destroy NADP : cells are suspended in 50 mM NaOH - glycine buffer pH 10.5 at 100 "C for 2 min, then 3 min at 60 "C and cooled. Cells are removed by filtration. This process eliminates NADP without loss of NADPH. On the contrary, NADP is extracted in 50 mM HC1- glycine buffer pH 3.0 for 3 min at lOO"C, then 3 rnin at room temperature and cooled. NADPH is totally

destroyed and NADP remains intact. NADP and NADPH are then estimated by the recycling method of Lowryet al. [34] except that the absorbance at 340 nm is measured (Gilford spectrophotometer).

Glutamine is extracted by boiling water for 5 min and estimated by glutaminase (Sigma Grade V) : 0.20 unit enzyme in 1 ml final volume 0.05 M phos- phate-citrate pH 4.9 for 90 rnin at 37 "C, followed by NH; determination as above.

RESULTS

Arginase Synthesis as a Response to the Presence of Substrate and the Need for Its Degradation

Arginine can be used as nitrogen nutrient by S. cerevisiae. The adaptive utilization of arginine is expressed by the increase of arginase synthesis when cells are grown on medium M with NH:, with NH; + arginine and with arginine respectively (Table 1, Expts 1 - 3). The presence of arginine stimulates arginase synthesis, but by far the largest increase occurs when arginine is the only nitrogen nutrient, i.e. in the absence of NH;. As one may expect from the comparison of the pathways of nitrogen assimilation, N H > is a better nutrient than arginine (Table 1, Expts 1,3). Hence, arginase synthesis appears to be logically promoted by two physiological conditions :

Table 1. Growth rate, pool und enzyme activities qf the wild-type strain C1278b a-Glucosidase activities are those reported in [19]

Expt Medium supplement with vitamins and Generation Arginase z-Glu- Pool no. ~- ~ time at cosiddse ~

carbon nutrient nitrogen nutrient 29 C arginine NH: glutamine

1 2 3 4 5

6 7 8 9

10 11 12 13

14 15 16

Glucose Glucose Glucose Glucose Glucose

Galactose Maltose Glucose Lactate Glucose Glucose Glucose Glucose

Glucose Glucose

min

NH; 120 NH: + arginine 120 arginine 150 ornithine 240 chemostat limited 170 b y N H > 700 NH; 130 NH: valine NH: > 600 glutamate 150 serine 165 urea 180 NH: + a,y-diaminobutyrate

- (200) 120

glutamine 120 asparagine 120

- (1000) 135

pmolx h - ' x mg protein-'

nmol/mg dry wt cells

6 0.4 80 30 49 20 300

250 760 160 35 5 35 < 1 7 10 8

63 0.4 30 to 40 70 50 6 110 20 20

40 95

10 27 8 23 150

Eur. J. Biochem. 4N (1974)


the presence of the substrate and the need for its degradation.

Such a situation suggests a mechanism which in- volves two components : a process of induction which transmits the signal of the presence of substrate, and a second device which might either control the entrance of inducer into the cell or allow the recog- nition of the presence of a “good nitrogen nutrient” inside the cell. The two processes might play simultaneously.

S. cerevisiue is endowed with the possibility of a control of inducer uptake by NH:. The rate of uptake of arginine (and most amino acids) is increased in the absence of NHf, in the growth medium, due to the activation of a non-specific (general) amino acid permease which by contrast to specific permeases, has a catabolic function [30]. Indeed the absence of NHf, provokes a 2.5-fold increase in the arginine pool (Table 1, Expts 2,3). That arginase synthesis does not run in parallel with accumulation of arginine does not exclude such control because compartmentation in eucaryotic cells may distort a simple correlation between the total pool and inducer concentration at its site of action. It is known that amino acids may be strongly and unequally concentrated in such a com- partments as the vacuole versus the cytosol[35]. In addition, an induction might occur at high concentration as a result of a rather limited increase because of a cooperative effect of inducer.

However a process independent of inducer exclusion is suggested by a number of observations. Nitro- gen starvation [36], growth in chemostat limited by NHf, and growth on nitrogen nutrients unrelated to arginine metabolism are able to promote arginase synthesis [15,18] (Table 1, Expts 5,10- 12 versus 1). Such experiments show that a part of the control is independent of the presence of external inducer. How- ever as they modify the rate of growth, they might simultaneously several factors such as the internal balance between inducer and repressor, the general biosynthetic capacity of the cell and its gross compo- sition. As useful as they can be in a first approach, such experiments cannot identify a specific regulatory process and call for a more rigorous approach.

The Influence of the Quality of the Nutrients on Arginase Synthesis

Arginine can be used as sole nitrogen nutrient, but it does not support growth as carbon nutrient. This agrees with the observation that glutamate itself, the end product of arginine catabolism, cannot be used as carbon nutrient. This is at variance with the situation in some other fungi and many bacteria where arginine and glutamate can serve as both carbon and

nitrogen sources. In some such cases it has been shown that the degradation of these amino acids is also under the control of the carbon nutrition [S, 13,371.

The synthesis of arginase is not significantly affected when galactose or maltose replaces glucose as carbon source. This is in contrast to the effect on a typical enzyme of carbon catabolism such as a-glucosidase (Table 1, Expt 6 versus 1). Galactose supports a rate of growth similar to glucose. Conversely the use of valine instead of NH: which reduces the growth rate of S.cerevisiae and stimulates the synthesis of arginase, does not affect a-glucosidase synthesis. From this it is tempting to generalize and to distinguish carbon and nitrogen catabolite repression. However on lactate, which is a very poor carbon source, there is a strong derepression of arginase, which is especially striking since the NH: pool is very high (Table 1, Expt 9). We shall return later to this apparent contradiction, but is is clear that carbon nutrition may affect arginase synthesis.

S.cerevisiae can grow with a great variety of nitrogen nutrients; in most cases the rate of growth is reduced when compared to NH:. This includes glutamate, arginine, proline, valine, urea, ornithine, citrulline, allantoin, etc. The only nitrogen nutrients which with our strain, support comparable rate of growth as NH: are glutamine and asparagine: under these growth conditions the level of arginase is similar and low (Table 1, Expts 15,16). One of these compounds might be the true metabolic signal which limits arginase synthesis. By using the binary nitrogen method of Thorne[38] we reached the same conclusion as this author: asparagine and glutamine are used preferentially to NH;. The addition of glutamine strongly reduces NH: utilization (Fig. 3) and reci- procally when NH; is added to cells growing with asparagine or glutamine respectively, NH is not utilized in the first case and poorly in the second if we compare to similar experiment with glutamate.

As a first attempt to identify the most likely metabolic signal, the pool of NHf, and glutamine have been measured when cells were grown on anyone of these nutrients compared with cells grown on several media. From the data reported in Table 1, it is clear that, in the absence of inducer and with glucose as carbon nutrient, arginase synthesis is low when NH; reaches a pool above 20 nmol per mg dry weight (Expts 1,15,16) and is promoted by a factor of 4 to 8 when NH: decreases (Expt 10). It is worth mentioning that a severe limitation of NH: in a slow chemostat reduces the NH: pool much below 5 , however it does not promote any further arginase synthesis (Expt 5). As growth on glutamate produces a high pool of glutamine, in spite of arginase derepression, NH ‘4 remains the most likely metabolic


608 Nitrogen Catabolite Repression of Arginase in S. cerevisiae

3 1 0 0.200 0.400 0.600 0.800

Growth ,absorbance at 660nm

Fig. 3. Preferential utilization of glutamine as the source of nitrogen ,from a mixture of glutamine and NH:. Glutamine, 7 mM final concentration, is added at the point shown by

the arrow into a culture growing with 5 mM (concentration of NH; at time 0) (O--O). ( x ~ x ) Culture on NH; without addition of glutamine

2 1

0 0.500 1.000 1.500 2.000 2.500 Growth , absorbance at 660nm

Fig. 4. Utilization of NH; in the presence oj duerent additions. 5 mM glutamine ( 0 4 ) ; asparagine (-0); aspartate

signal in spite of not being the one taken preferentially by the cells (Expts 1,lO).

The Influence of Nitrogen Nutrition in Mutants Affected in the Process of Induction

As mentioned in the Introduction the process of induction can be lost completely as a result of two types of mutations [15]. Mutations at the operator site for arginase such as carg+O--2 provoke a very high level of arginase, but this level remains unchanged when arginine is added to medium M with NHZ (Table 2, Expts 9,lO versus 1,2). However when NH; is excluded from the medium and replaced by glutamate, or when NH; is limited by chemostat, an additional synthesis of arginase occurs. This shows that the stimulation of arginase production observed by nitrogen limitation in wild type is at least partially retained when the operator is inoperative with respect to arginine induction (Table 2, Expts 12,13, as 8 ,5 ) .

One reaches the same conclusion with argR- mutants in which the initial step of induction, the

(x-x); glutamate (0-0). NH; is added at the arrow at 2 mM final concentration

input of the induction signal is lost. argR- mutants have half the arginase level of wild-type cells growing on medium M with NH: and addition of inducer does not modify this very low arginase synthesis (Table 2, Expts 15- 17 versus 1,2). However, conditions of nitrogen limitation stimulate arginase synthesis by a factor of 4 to 9 as in wild-type cells (Expts 19-21 versus 15 to be compared with Expts 5 , 7,8 versus 1). Hence one may conclude that at least a part of the stimulation of arginase synthesis by nitrogen limitation is completely independent of the initial step of induction. We may call this effect a nitrogen catabolite repression, currently expressed by the reduction of arginase synthesis when a good nitrogen nutrient is added to medium of lower nitrogen nutrition capacity.

It remains that exclusion of inducer may be an important device which limits arginase synthesis in the wild-type strain when the induction mechanism is intact. Indeed the high level of operator mutation leaves a large capacity for an induction exceeding what is observed by addition of arginine to medium 150



Table 2. Arginase activities in induction mutant

Expt Genotype, name of strain Nitrogen compounds added Generation Arginase activity no. to medium M time at 29 "C

1 2 3

4 5 6 7 8

9 10 11 12 13 14

15 16 17 18

19 20 21 22

23 24 25 26 27 28 29

30

31

min

Wild-type (Z32278b)

cargA+O--Z (7204b) arginase operator constitutive

argR- II-10 (BJ210) non-inducible

gdhA --I (4324~) lacking glutamate dehydrogenase A

gdhA--l, argR-II-I0 (0315b)

gdhA--l, cargA'0--2 (0311a)

NH: NH: + arginine NH: + glutamate

arginine glutamate serine proline chemostat NH: limiting

NH' NH ' + arginine NH: + glutamate chemostat NH; limiting glutamate arginine

120 120 120

150 150 165 180 170

NH+ 120 NH 2 + arginine NH' + DL-cc,y-diaminobutyrate (1 00) NH: + glutamate

glutamate proline chemostdt NH: limiting arginine

NH' N H ~ + glutamate NH? + glutamine N H ~ + asparagine proline NH: + arginine arginine

NH: + glutamate

NH: + glutamate

> 480

210 120

pmol urea x h-' x mg protein

6 20

8

250 50 20 25 35

138 136 129 222 230 350

3.6 3.5 4.8 5.8

33 16 15

40 35 50 34 23

350 350

17

185

with NH: and a gratuitous inducer, a,y-diaminobutyrate is a better inducer than arginine (Table 1, Expts 13,14 versus 2).

The Release of the Nitrogen Cataholite Repression by the gdhA- Mutation

It is well known that yeast and probably most fungi have two glutamate dehydrogenase working respectively with NADP and NAD [39,40].

Mutations and conditions of growth leave no doubt that the NADP-specific enzyme glutamate dehydro-

genase (NADP) has an anabolic function being the first step in the assimilation of NH:. Mutation gdhA- located in the structural gene of glutamate dehydrogenase (NADP) reduces the rate of growth; the residual growth can be due to the NAD-specific enzyme. This enzyme glutamate dehydrogenase (NAD) is a catabolic enzyme: it is present in strongly reduced amount under conditions which do not oblige the cell to deaminate glutamate, such as in the presence of NH:, and gdhCR mutation which restores normal growth on NH: provokes a high permanent produc-



Table 3. Pool qfamino acids irz wild-type (212786) and gdhA- mutant ( 4 3 2 4 ~ )

Growth in medium M with

NH: proline proline + NH: glutamate + N H ~ , glutamate

C1278b 4324c C1278b 4324c C1278b 4324c C1278b 4324c C1278b

nmol/mg dry wt cells

Alanine Arginine Aspartate Asparagine-glutamine Citrulline C ystathionine Glutamate Glycine Histidine Isoleucine Leucine Lysine Methionine

Ornithine Phenylalanine Proline Serine Threonine Tyrosine Valine

NH;,

36 55 27 41 29

115 18

0.9

4.8 2.8 2.3

15

35 26 0.5 0

17 18

-

0.7 9.7

17 37 15 27

1.4 0.3

4.8 2.2 1.6 1.8 6.9 0.2

13.6 0.5 0 8.2 8.6 1 .o 5.1

69

185

34 16 10 37 1.8 0.6

166 14 1.6 1.7 1.2

15

13 3.4 0.1

213 11 8.5 0.4 5.4

55 32 16 56 2.5 0.8

276 13 3.7 3 2.6

22 0.6

34 18 1

224 15 14 1.4 8.3

29 61 24 42 14

78 12 6 2.1 1.6

1.3

10

24 15

-0 14 14 0.4 7

-

0.4

41 42 74 102 14 24 91 33 12 7

137 200 15.6 17 5.7 7 2.4 2.5 1.9 2.5

2.6

0.9 1.3

12.4 34

141 39 33-46 33

-

0.4 0.8 51 15 20 1 1 18 0.5 1.2 6.5 10

20 36 11 97 11

183 12 5 2.3 2.3

-

20

219 48

-

- -

22 10 -

7.8

35 93 13

104 15

297 21

8 3 4

26

38 48

-

-

-

41 20

11 -

tion of glutamate dehydrogenase (NAD) which may compensate for the lack of glutamate dehydrogenase (NADP) [19].

In bacteria, NH: assimilation may occur by the succession of glutamine synthetase and glutamate synthase [41]. The yeast Schizosaccharomyces yombe has a NAD-specific glutamate synthase [42]. Recently Roon et al. have found a similar NAD-dependent glutamate synthase in S. cerevisiae, opening the possibility that this enzyme might be responsible for the residual growth of gdhA - mutants [43]. This, as an alternative or an additional compensatory process of gdhA mutation, is under investigation.

As expected, ghdA- mutants grown on medium 150 with NH '4 have a reduced amino acid pool (Table 3). This is due to the low production of glutamate. A normal pool as well as a normal growth rate is resumed by addition of glutamate or proline (Table 3 and Table 2: Expt 24 versus 23).

The interest of the gdhA mutation arose from the early observation that this mutation releases the in- hibitory effect of NHf, on the general amino acid- permease and that the effect of the mutation is not restored when the abolition of the catalytic activity is compensated by addition of proline [17].

These results can fit in the general view that inhibition by NH: is a nitrogen catabolic control, the general amino acid permease having a catabolic function. This has been further demonstrated when it was shown that the gdhA - mutation releases the NH: effect on the synthesis of at least three nitrogen catabolic enzymes : arginase, allantoinase and urea amidolyase [17,18].

Although it is clear that the NH:effect was released in the absence of inducer and in spite of the metabolic compensation by glutamate (Table 2, Expts 23,24), a more rigorous proof that the high level of arginase is not due to secondary effect was needed. It can be given by the genetical combination of the gdhA- mutation and the two mutations affecting induction.

In Table 2, it can be seen that the gdhA- mutation derepresses arginase synthesis in both argR-, non- inducible mutant (Expt 30 versus 18) and operator constitutive (cargA' O-) arginase mutants (Expt 31 versus 11). In both cases, the gdhA- mutation stimulates the effect of nitrogen limitation (Expt 30 versus21, 31 versus 12).

On may conclude that the gdhA- mutation suppresses the nitrogen catabolite repression of arginase indepedently of its effect on permeability or of its effect on growth rate.

Eur. J . Biochem. 48 (1974)

E. Dubois, M. Grenson, and J.-M. Wiame 61 1

Table 4. Pool of substrates and products of the glutamate deehpdrogenase A GDHase A, glutamate dehydrogenase (NADP)

Expt. Genotype, Nitrogen compounds Enzyme activity Pool no. name of the strain added to culture

medium M arginase GDHaseA NH', 2-oxoglu- NADP NADPH tarate

6 I

8 9

10 11

Wild-type (C1278b) NH+ N H ~ + glutamate glutamate valine NH: but lactate

replaces glucose

gdhA -- (4324~) NH' NH< + glutamate

Aconitase mutant (MG1748)

NH+ NH: glutamine asparagine

pmol x h-' x mg protein-'

nmol/pg dry wt cells

6 50 8 37

55 36 63

30-40

40 35

75 45 19 35 12 56

30 20

5

70

21 5 220

235 48

1.6 0.320 0.500 0.340 0.540

0.260 0.500 12

0.5

1.4 4.6 0.350 0.340

0.4 0.9 1.3 0.4

a Since growth on lactate provokes clumps, the dry weight was effectively measured in this case. A value of 0.3 is the minimal value which can be appreciated.

The Mechanism of the Participation o j the Anabolic Glutamate Dehydrogenase in Nitrogen Catabolite Repression

Whatever be the succession of events between addition of NH: and repression of a limited number of genes, a metabolite such as NH: itself or a derivd- tive must find a specific receptor which transforms the metabolic signal into a genetical one. The first receptor does not necessarily act directly at the genetical level. The participation of the glutamate dehydrogenase (NADP) to the control allows in a first stage to eliminate a number of possibilities.

It was already mentioned that addition of glutamate restores a normal growth and a normal pool of amino acids in the gdhA - mutants without repression of arginase, allantoinase and urea amidolyase. Hence glutamic acid cannot be the derivative of NH: which acts by itself as the metabolic signal for repression. This was already suggested as growth on medium M with glutamate provokes a derepression.

Among other likely derivatives, glutamine and asparagine can also be eliminated: addition of these amino acids does not restore repression in the mutants. In agreement with that, we have seen that in cells grown on medium M with glutamate the pool of glutamine is higher than in cells grown on medium M

with NH: (Table 1, Expt 10 versus 1). In the gdhA- mutant, the pool of NH; is greatly increased [17] (Table 4). The gdhA - mutation resides in the structural gene for glutamate dehydrogenase (NADP). The possibility of another mutation strongly linked to gdhA- and affecting independently the nitrogen catabolite repression is excluded because a number of gdhA- mutants have been obtained as growing slowly on NH;, i.e. without taking advantage of the nitrogen catabolite derepression [19]. So there is no reason to believe that the receptor of NHf, is damaged by another mutation. If such receptor exists it can only be the glutamate dehydrogenase (NADP) itself. As the glutamate dehydrogenase (NADP) has other substrates and products than NHf, and glutamate, the mutation could affect the concentration other products acting secondarily as signal. In wild-type cells, due to the catalytic function of the enzyme, NADP could increase and NADPH could decrease. Results reported in Table 4 show that, if any change occurs, it is not in that sense and the gdhA- mutants have the same pool as the wild-type cells (Expt 7 versus 1). Thus these two compounds cannot be considered as effectors. The last compound which remains to be considered is 2-oxoglutarate. The pool has been measured in the wild-type strain, in the gdhA- mutant and in the



mutant MG1748 which is affected in its aconitase activity and hence should have a depressed pool of 2-oxoglutarate. By itself the 2-oxoglutarate cannot be the only effector because the g d h K mutant has the same pool as the wild type when both are growing on medium M which NH: (Table 4, Expt 6 versus 1). In the aconitase mutant growing on medium M with NH; one observes a strong arginase derepression. The NH: pool is very high and the 2-oxoglutarate is the lowest which has been observed (Table 4, Expt 8 versus 1). As the glutamate dehydrogenase (NADP) is normal, one must conclude that NH; + glutamate dehydrogenase (NADP) is not a sufficient combination for repression and that the complete signal for repression could be a combination of the enzyme with both substrates at a level such as the one found in wild-type cells growing on NH:.

Let us analyze such a proposition. In what concerns nitrogen catabolite repression, the aconitase mutant can be repaired by proper addition of the medium. This is fundamentally different from the g d h K mutant. In other words, the aconitase itself does not act by its mere presence but by its catalytic activity. When glutamine is the nitrogen source, arginase is repressed at almost the wild-type level. The level of 2-oxoglutarate is slightly lower than normal (by normal we mean as in wild-type strain growing on medium M with NH:) (Table 4, Expt 10).

Addition of glutamate to medium 150 with NH: (Expt 9) partially restores the repression, the 2-0x0- glutarate pool is half normal, the NH; pool is high. Medium M with asparagine provokes a strong derepression with a high NH i pool and the lowest level of 2-oxoglutarate (Table 4, Expts 9 , l l ) . From these data it is tempting to propose that NH: and 2-0x0- glutarate need to cooperate at the level of glutamate dehydrogenase (NADP) to provoke repression. The glutamate dehydrogenase (NADP) would be the receptor of a double metabolic signal, NH: + 2-0x0- glutarate. In the absence of glutamate dehydrogenase (NADP), even with both high NH; and 2-oxoglutarate such as in medium M with NH: + glutamate, there is no repression.

From this, we may return to the unexpected arginase derepression by growth on lactate + NHf,. As expected, the NH; pool is high, but the 2-0x0- glutarate is at the lowest level (Table 4, Expt 5). The 2-oxoglutarate in this case may be the effector which is lacking for arginase repression.

As our proposition leads to separate the catalytic and the regulatory functions of the glutamate dehydrogenase (NADP) one could expect mutations of glutamate dehydrogenase (NADP) which would independently affect each function. One can foresee that such a situation may occur especially if the two sub-

( 4 ) 514144

T ( 3 ) MG1749

ED 220

50821 a

ED 300 k 0 0 20 40 60

( p o l ' h - ' . mg protein-')

Fig. 5. Arginase activity as a junction of activity of glutamate dehydrogenase ( N A D P ) , after growth on medium 150 with N H f , + glutamate. See Materials and Methods for the strains. T are tetraploids containing 4 (T4) to 0 (TO) mutated genes. C1 is a diploid (gdhA--l xgdhA--5) with an intracistronic complementation. Other strains are mutants or revertants of MG1694 described earlier [I91

Activity of glutamate dehydrogenase A

strates NH '4 and 2-oxoglutarate have regulatory sites separated from the catalytic ones. However our results, on the contrary, show a striking parallelism between the catalytic activity and the regulatory capacity in a large number of combinations (Fig.5). The regulatory function does not scatter when plotted as a function of the catalytic one. The whole regulatory function is obtained when the catalytic activity reaches about 10% of its normal value and this is true in the case of gene dosage in polyploids as well as in haploid revertants and in intracistronic complementation lead- ing to partial recovery of activity. As the data presented above do not indicate a participation of NADP, NADPH or glutamate, the stringent linkage of the regulatory and of the catalytic functions speaks in favour of an identity for the regulatory and the catalytic sites.

Behaviour of Strains of Different Origins

Wild types of different origins behave differently. This is not surprising as yeasts are really domesti- cated organisms selected by man over a long time of



Table 5. Ejject of N H f , and nitrogen nutrients on permease activity and enzyme synthesis for different wild-type strains GDHase A, glutamate dehydrogenase (NADP). Amino acid permease activity expressed as uptakeof nmol of tryptophan x mg protein-' xmin-'

Expt. Nitrogen nutrient added Strain Origin no. to medium M

General amino Enzyme activities acid permease activity Arginase GDHase A

pmol x h-' x mg protein-'

2 C1278b [I61

1 2 3 4

5 6 7

8 9

10

11 12 13

14 15 16

17 18 19 20

21

NH' NH: + glutamate Glutamate NH', + casamino

Casamino acid (1 x) Complex medium 863 Proline

acid (1 %)

6 50 8 37

55 36

27 40 26

20 102 4324c gdhA--1 from C1278b

~ 7 1 40 20 55

NH: Proline Glutamate

88 92

S288c 12

96

18

9 22

NH', Glutamate Proline

H 1326 Hutter NH: Glutamate Proline

9 23

100

16 FLlOO

6 63 17 29

N H ~ , Glutamate Proline Complex medium 863

134 5

RD13-sp23 gdhhA--11 derivative [I91 of FLIOO

NH', + glutamate 17

fermentation practice. It was mentioned earlier that the level of an enzyme like ornithine carbamoyltrans- ferase may change as much as six-fold from strain to strain, which points to the need of isogenic partners in the quantitative genetical analysis of enzymes synthesis [16]. Our strain C1278b is especially useful for the study of NH: repression as well as of NH: inhibition on the general amino acid permease. In Table 5 we compare wild-type strains commonly used in different laboratories. The NH; effect on the general amino acid permease is judged by comparing tryptophan uptake when cells are growing on medium M with NH: and proline, respectively. Derepression of arginase by growth on medium M with glutamate to be compared with growth on medium M with NH: + glutamate or medium M with NH: reaches a ratio of 9 in the wild-type strain C1278b and a ratio of 3 with FLlOO (Table 5, Expts 1,3,17,18). As expected the gdhA- derivative of FLlOO shows only a three-fold

derepression (Table 5, Expt 21). Another difference concerns repression of the glutamate dehydrogenase (NADP) by growth in complex medium[44]. Strain FLlOO is repressed by a factor of 10 [25] compared to a factor of 2 in C1278b (Table 5, Expts 1,6,17,20).

Nitrogen Starvation as a Tool to Study Catabolite Repression

In Table 6 we report results on the effect of total nitrogen starvation on arginase synthesis. We did not use such procedure in other sections, because it ob- viously may introduce secondary effects not directly related to the mechanism of the NH; effect. However recent works of Whithey et al. [45,46] led us to in- vestigate a few cases because some of the results of these authors appear contradictory with what is presented here and in previous works [l, 151.

In Expt 5 it is shown that the non-inducible argR- mutant, when starved, does not produce more ar-

Eur. J . Biochem. 48 (1974)


Table 6. Effect of starvation on arginase synthesis Enzyme activities expressed as rate of formation of product

Expt Genotype no.

Strains Nitrogen nutrient Enzyme activities and addition

urea amidolyase arginase after starvation of

O h l h 2 h ~~~ ~~

1 Wild-type Z1278b 2 Wild-type 3 Wild-type 4 cargA (arginase deficient) AG2 5 argRII-- 10 BJ210 6 urgC- (phospho-acetyl-

glutamate dehydrogenase MG690 deficient)

nmol x h-’ x mg protein-’

pmol x h-’ x mg protein-’

~ ~~

NHf, I x 7 13 87

NHf, + glutamate

NH: 3 3 3

glutamate 30 x 10-l 1 x 10-1

glutamate 30x

NH’, + 20 pg ornithine 12 12 15

ginase, this is not expected since we know that we could promote arginase synthesis in the same mutant either by growth on some other nitrogen nutrients than NH; such as glutamate, or by limiting NH: in a chemostat or by introducing a g d h K mutation in an argR- mutant (see Expts 19 - 21,30 of Table 2). So starvation which at first view is expected to be a more drastic tool than NH: limitation does not provoke the release of NH; effect. We confirm the finding of Whitney et al. that at the opposite to wild type (Expt l), an arginine auxotroph previously grown with a limited amount of ornithine is not derepressed by nitrogen starvation (Expt 6) unless an inducer is present [45]. In that case, it is difficult to use exponential growth because of auxotrophy. The same authors have also shown that starvation does not promote urea amidolyase in an arginine-less mutant, because, of the lack of urea formation. However in a similar strain, we obtain a strong derepression by growth on medium M with glutamate (Table 6, Expt 4).

These experiments show that the methods used are not equivalent. The different results are not due to peculiarity of strains. It is obvious that presence of inducer and intact argR gene are required for arginase synthesis when nitrogen starvation is total. In view of our previous finding that the inducer may act in two processes [l] and that starvation may introduce more uncontrollable secondary effects than normal growth, we feel more secure in following the general method- ology used in the present work, keeping in mind that, if understood, the contradiction may clarify a funda- mental mechanism.

CONCLUSION AND DISCUSSION

We conclude at least a part of the NH: effect on arginase synthesis is independent of substrate induction. This is established by showing that NH: effect is retained in mutants which are no longer inducible by endogenous or exogenous arginine, and in fully constitutive operator mutants. This process, which may be named “nitrogen or ammonia catabolite repression”, is lost as a result of the gdhA- mutation. As expected on the basis of the above conclusion, this mutation derepresses arginase also in non-inducible and in fully constitutive mutants.

The gdhA ~ mutation affects the structural gene for glutamate dehydrogenase (NADP). This makes sense as this enzyme is the first enzyme of the NH assimilatory pathway, and when supplied with its substrates it makes superfluous the degradation of an elaborate nitrogen nutrient such as arginine.

It was already known that glutamate compensates for the metabolic defect of the gdhA- mutation but does not restore enzyme repression [18]. All other additions, including the best nitrogen nutrients for yeast, glutamine and asparagine, do not restore ammonium repression either. As the pool of NADP or NADPH does not change from condition of derepression to repression, the enzyme itself appears to be a regulatory protein independently of its metabolic activity. In addition, as the amount of glutamate dehydrogenase A does not change from a state of repression (NH: + glutamate) to derepression (glutamate, Table 4) it must act by receiving metabolic signals. Although conditions of derepression are usu-



ally correlated with a low pool of NH:,, in a mutant with reduced aconitase activity (and with an intact glutamate dehydrogenase A), derepression occurs in spite of a high NH; pool. This can also be provoked in wild type with a poor carbon nutrition. As opposed to gdhA - mutants, aconitase mutants may recover repression if the nutrition allows the formation of a normal pool of 2-oxoglutarate and NH:. It is proposed that these substrates of glutamate dehydrogenase (NADP) are the metabolic signals and must reach simultaneously a minimal value to transfer the signal of repression through a change of the physical con- formation of the glutamate dehydrogenase (NADP). Although the nitrogen repression is usually independent of sugar nutrition, the participation of 2-0x0- glutarate to this regulation explains the derepression provoked by lactate and may be the cause which leads to the well known interaction between carbon and nitrogen catabolite repression (see [38,13]). In the present work the level of glutamate dehydrogenase (NADP) has been verified to remain unaffected for instance in the aconitase mutant growing on NH: (Table 4, Expt 8). Of course other conditions which would could inactivate or repress glutamate dehydrogenase (NADP) as observed in Aspergillus might result in derepression [37].

It is likely that glutamate dehydrogenase (NADP) plays the same role in the regulation of allantoinase and urea amidolyase in yeast [18] as well as of some nitrogen catabolite enzymes of Aspergillus niduluns as shown by Arst and McDonald [20] and confirmed in Pateman’s laboratory [21].

However the conclusion of these two laboratories are divergent. Arst and Cove [37] have presented evidence for the occurrence of a genetic locus ureA which can be interpreted as a gene coding for a protein necessary (positive control) for the synthesis of a large number of nitrogen catabolite enzyme without affecting ammonia assimilation. This is an obvious important progress in the field of nitrogen catabolism. They consider that glutamate dehydrogenase (NADP) does not act “directly” (quotation marks introduced by us) in the regulatory mechanism but by a process such as a modification of the intracellular distribution of ammonium. Their main reason is that it is unlikely that a protein which is an enzyme could act in such a variety of sites as to recognize not only its substrates but also to inhibit so many processes as permeases and proteins such as the one coded by ureA gene.

Our view presented here and summarized some time ago in a symposium [l] is that the glutamate dehydrogenase (NADP) is the true receptor of NH: for a limited number of nitrogen catabolic activities. It is worth recalling that a few years ago, the possibility that an enzyme could act as a reversible effector of

another enzyme was unknown, although today, this is proved without doubt [24]. The regulation of the synthesis of nitrogen catabolic enzymes in Klebsiellu uerogenes appears to be mediated by the molecules of glutamine synthetase independently of their catalytic function [47,48], and the threonine deaminase appears to be involved in the regulation of isoleucine and valine biosynthesis [49,50]. See Goldberger [51] for a review.

When compared with simple protein-metabolite interactions, protein-protein interactions add another scale of refinement in biological processes mainly for two reasons: the cumulation of the specificity of the two interacting proteins and the occurrence of allo- steric transition which brings the regulatory system in a state of cooperativity in the proper range of con- centrations of metabolites compatible with the catalytic activity of the enzyme which plays at the same time a regulatory role.

Pateman and collaborators favor the view that the glutamate dehydrogenase (NADP) is directly involved in NH ‘4 repression [52]. Their experimental analysis is still too limited to add further precision to what has been expressed elsewhere [17,18, I].

If induction and nitrogen repression were the only and totally independent regulatory circuits of arginase synthesis, addition of dghA - mutation and operator constitutive mutation for arginase (curgA + 0- ) should give the maximal rate of synthesis. Expt 31 of Table 2 compared with Expts 14 and 29 shows that this is not the case; cells with either a gdhA ~ mutation alone or cargA+O- alone, growing on arginine as sole source of nitrogen have a higher level of arginase than the double mutant growing with NH: + glutamate. This indicate that an additional mechanism is involved [l 1.

E. D. is Aspirant du Fonds Nutional de lu Recherche Scientijkpe. We are grateful to Drs Arst and Cove as well as Dr Roon for prompt communication of the results of their work. This work was supported by contract no. 985 from the Fonds de la Recherche Fondanzentale Collective.

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E. Dubois, M. Grenson, and J.-M. Wiame, Institut de Recherches du C.E.R.I.A., Avenue Emile-Gryzon 1, B-1070 Bruxelles, Belgium
