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

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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 luniversite Libre de Bruxelles, and Institut de Recherches du Centre dEnseignement 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 addi- tion 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-gluta- mate : 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-histi- dine 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 con- ditions are required for the full promotion of the synthesis of an enzyme which belongs to such a path- way [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 physio- logical 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 in- volved 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 syn- thesis 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, orni- thine 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 cata- bolic enzyme, the NAD-specific glutamate dehydro- genase, does not loose its NH; control as a result of thegdhhA- mutation [17- 191.

    From these works it was concluded that the ana- bolic 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 effec- tor(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) ac- tivity 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 cere- visiae [ 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.

    Eur. J. Biochem. 48 (1974)

  • 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 ar- ginase [ 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].

    Eur. J. Biochem. 48 (1974)

  • 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 0C 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, elec- trode (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 assimila- tion, N H > is a better nutrient than argini...

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