On the Nature of argR Mutations in Saccharomyces cerevisiae

Eur. J. Biochem. 43,87-92 (1974)

On the Nature of argR Mutations in Saccharomyces cerevisiae

Philippe P. HOET and Jean-Marie WIAME Laboratoire de Microbiologie de l’Universit6 Libre de Bruxelles, and Institut de Recherches du Centre d’lhseignement et de Recherche des Industries Alimentaires et Chimiques, Bruxelles

(Received November 7, 1973)

Ornithine, citrulline and homoarginine having a regulatory role on arginine biosynthetic enzymes in wiId-type, but not in argR mutants, do not interfere with aminoacylation of tRNAArg in vitro or in vivo. The same aminoacylation reaction does not seem to be modified in argR mutations, strengthening the hypothesis of a mutation, involving the apopressor.

Increasing evidence for the participation of activated amino acids in the process of repression has accumulated over the past years [l-61. More recently, arginine has been included in such a class in the case of Escherichia coli [7,8]. The behaviour of some mutants of Neurospora crassa [9] and Chlamydomonas reinhardii [ 101 have been interpreted in the same way. If such is the case most character- istics of mutants taken as regulatory mutants affecting an aporepressor in a negative type of control would be simulated by mutations of the activating enzyme or by mutations affecting the synthesis or the structure of aminoacyl-tRNA.

argR- mutations of Xaccharomyces cerevisiae may be interpreted as mutations affecting the pro- duction of the aporepressor involved in the repression of the arginine biosynthetic pathway. These mutations cause derepression of some of the biosynthetic enzymes, when cells are grown on minimal medium, and these enzymes are no more repressible in the presence of arginine. The rate of growth is not affected. These mutations are recessive and do not show linkage with the structural genes of the enzymes on which they act. The permeability for arginine and the pool of this presumed corepressor is not reduced by these mutations [II, 121.

The nature of the element affected by these mutations is of particular interest because of two unexpected properties which may be peculiar to regulatory mechanisms in an eucaryotic organism, for which no counterpart has been described so far in procaryotes. argR- mutations belong to three classes argRI-, argRII-, argRIII- which behave similarly

Enzymes. (CBN Recommendations, 1972) L-Ornithine transcarbamylase or carbamoylphosphate : L-ornithine carb- amoyltransferase (EC 2.1.3.3) ; argininosuccinase (EC 4.3.2.1) ; arginyl-tRNA synthetase or L-arginine : tRNA*rg ligase (AMP-forming) (EC 6.1 A.19).

and are distinguished only by complementation and absence of genetic linkage. If they affect the synthesis of an aporepressor, this compound should have an heteropolypeptidic structure. I n addition to their influence on the synthesis of anabolic enzymes these mutations affect, but in the reverse way, the synthesis of the catabolic enzymes, introducing the possibility that concurrent pathways, biosynthesis and degradation, of a given compound, can be under the control of a repressor with two separate functions

The alternative could be that arginine is neither the true corepressor of the biosynthesis nor the true inducer of the degradation. Corepressor and inducer however are identical because genes argRI, argRII and argRIII similarly affect the catabolic pathway. I n this case, arginyl-tRNA is presently the only candidate for such a function. Either alternative hypothesis is of an obvious interest.

A way to investigate this problem was suggested by recent work, showing that arginine is not the only compound leading to regulation of both biosynthetic and catabolic enzymes [13]. Homoarginine, ornithine, 2,4-diaminobutyrate and lysine act in the same way and their action is cancelled by argR- mutations. Since all these compounds could hardly be activated into acyl-tRNA, their eventual action on the aminoacylation of arginine remained to be investigated.

~ 3 1 .

MATERIALS AND METHODS Materials

~-(Guccnido-~~C)arginine, 30-45 mCi/mM, was obtained from CEN (Mol-Donk, Belgium). Arginino- succinic acid was from Sigma Chem. (St. Louis, Mo.). tRNA (brewer’s yeast) was from Schwarz Bioresearch Inc.

Em. J. Biochem. 43 (1974)

88 argR Mutations in Saccharomyces cerevisiae

Strains of Saccharomyces cerevisiae and Culture Media

The mineral base for all growth media 1111 con- tained no nitrogen. Only the added nitrogen source will be designated (see Table 1). Glucose as carbon source and vitamins were added as described [ll]. Cultures were grown on a rotary shaker a t 30 "C. A portion of the mutant culture was taken before killing and checked for their phenotype.

The mutant strains used are isogenic to wild-type X. cerevisiae 2 1278 B (a) . The regulatory mutant bearing the mutation argRI-2 (BJ 102) has been described [ll]. The argininosuccinase (argH-) defi- cient mutant (MG542) is described in Hilger et ail. ~ 4 1 .

Extraction of t R N A tRNA was extracted according to Folk and Berg

1151 with modifications described below. 200 ml 500/, trichloroacetic acid were added to 2.5-1 cultures and 1 min later 20 ml lo/, sodium dodecylsulfate. The mixture was swirled for 15min, then chilled, centrifuged and stored at -20 "C. For tRNA extraction, the cells were resuspended in 100 ml 0.25 M sodium acetate buffer (pH 5.4) containing 1 mM EDTA and 0.050/, sodium dodecylsulfate.

An equal volume of phenol, saturated with 50 mM sodium citrate buffer (pH 5.0), was added and shaken vigorously for 10 min a t room temperature. After centrifugation, the upper aqueous layer was syphoned off and after addition of 100ml phenol shaken for another 1Omin. After centrifugation, the aqueous layer was once more syphoned off, and extracted three times with twice the volume of ether. The two phases were separated and RNA was precipitated with two volumes of ethanol in the presence of 0.1 volume of 2001, potassium acetate. After standing for a t least 30min, the precipitate was centrifuged and the RNA resuspended in 6 m l 0.1 M sodium acetate buffer (pH 4.7). This was put on top of a DEAE-cellulose column, preconditioned with 50 mM sodium acetate buffer (pH 4.79, containing 0.25 M NaCl and 0.01 M MgClz. The column was thoroughly washed, before eluting the tRNA with the same buffer, containing 1 M NaC1. The eluate was precipitated with ethanol a t -20 "C. tRNA concentration is measured spectrophotometrically a t 260 nm (1 unit of absorbance = 40 pgglml tRNA).

Periodate Oxidation : Determination of the Percentage of Acylated tRNAArg in vivo

After centrifuging the extracted tRNA, the precipitate was resuspended in 6 ml 0.1 M sodium acetate buffer (pH 4.7). Periodate oxidation was done on half of this aliquot. The other half went through the same operations except for the addition of

periodate. The sample was incubated with sodium periodate ( 2 mM final concentration) for 30 min in the dark a t room temperature. After precipitation and centrifugation, the excess of periodate in the supernatant was ascertained by measuring the decrease in absorbance at 232 nm upon addition of a few drops of 1 M ethyleneglycol. The pellets were resuspended in 2 ml 0.1 M acetate buffer (pH 4.7), made 0.1 M in ethyleneglycol in order to remove the excess periodate. After precipitation and centrifugation, the samples were resuspended in 0.05 M sodium carbonate buffer (pH 10.2) and incubated for 20 min a t 37 "C in order to deacylate the tRNA. After dialysing the samples for a t least 3 h, the arginine acceptor capacity was determined for both periodate-treated and control tRNA. The fraction of periodate-protected acceptor capacity measures the extent of acylation of tRNA*rg in vivo. As a control of our method, arninoacylation was measured in vivo in a bradytrophic strain, suspected to have a lowered percentage of aminoacylation ; indeed, the fraction of aminoacylated tRNA**g was 37O/,, when arginine was omitted from the growth medium, and 6601, when arginine, added to the medium, restored the growth rate to normal.

Aminoacyt-tRNA Synthetase Cells were suspended in a buffer containing 0.1 M

Tris-HC1 (pH 7.4), 1 mM reduced glutathione, 1 mM MgCl,, and 60mM KCl. The cell suspension was lysed by passage through a French pressure cell, and then centrifuged first a t low speed to remove unbroken cells, and than at 35000 rev./min for 30min in a Spinco centrifuge. The supernatant liquid was passed through a 1 x 10-cm Sephadex G-25 column, equilibrated with the same buffer. 30°/, glycerol was added to this enzyme preparation and then stored at -20 "C.

Acylation of t R N A by Arginine: Conditions of Assay The following conditions were used: 70 pg tRNA,

1 pmol ATP, 1 pmol MgCl,, enzyme preparation corresponding to 20 pg protein, 7 pmol Tris-HC1 (pH 7.4) or 7 pmol sodium cacodylate (pH 7.1), 70 pmol reduced glutathione, 4.2 pmol KC1 and [14C]arginine (45 mCi/mM) were mixed in a total volume of 0.120 ml and incubated a t 37 "C for 20min. The reaction was stopped by adding cold l o o / , trichloroacetic acid containing 150 mg/l cas- amino acids. The precipitates were collected on nitro- cellulose (millipore) filters and washed thoroughly. The filters were then dried and their radioactivity was counted in a liquid scintillation counter. Using this method, Km for arginine of arginyl-tRNA synthetase is shown to be about 2.0pM. Since canavanine, analogue of arginine, is known to be incor-

Eur. J. Biochem. 43 (1974)

P. P. Hoet and J.-M. Wiame 89

porated into proteins, its inhibitory effect on arginyl- tRNA synthetase was examined, as control of our method. When adding canavanine, a competitive inhibition was seen, as revealed by plotting the results according to Lineweaver and Burk (see Fig. 2).

Peculiarities of the Aminoacylution Reaction When adding limiting amounts of enzyme, the

enzyme concentration determines not only the initial velocity of aminoacylation, but also the plateau value finally reached. These levels correspond to incomplete aminoacylations, recently studied in detail by Bonnet and Ebel [IS]. According to this study, incomplete aminoacylations result from a steady state between aminoacylation and enzymic and non-enzymic hydrolysis of the aminoacyl-tRNA.

Aminoacylation kinetics were studied as a function of enzyme concentration in our experimental conditions : 0.16 mM [14C]arginine and limiting amounts of cell-free extract (from 1.25 pg to 5.0 pg protein) were added to a reaction mixture, as described in the previous section. The reaction was started by adding ATP. Fig.1 shows that not only the initial velocity of the reaction, but also the plateau value, is determined by the enzyme concentration. We checked if incomplete aminoacylation could be due to deacylation of [14C]arginyl-tRNA in our experimental conditions. [14C]Arginyl-tRNA was synthesized in a reaction mixture, similar to the one described in the previous section, except that the quantities of the constituents were scaled up. The product of the reaction was isolated by adsorption to and elution from a DEAE-cellulose column. The eluate was precipitated and resuspended in different buffers. Deacylation occurred in the samples, where the pI1: of the buffer was above neutrality. The overall analysis of Bonnet and Ebel [16] thus seems applicable to our experimental conditions.

When measuring in our study the activity of arginyl-tRNA synthetase for arginine (and the products metabolically related to arginine) the amounts of aminoacyl-tRNA were measured a t their plateau value, corresponding to a steady state between a given rate of synthesis and deacylation of aminoacyl- tRNA. The plateau values thus obtained are a measure of this rate of synthesis.

RESULTS AMINOACYLATION OF tRNAArg in vitro : INFLUENCE O F PRODUCTS, AXFECTING THE REGULATION OF ARGININE BIOSYNTHESIS

Ornithine and Citrulline Ornithine and citrulline modify the level of orni-

thine transcarbamylase in a way that could not be correlated with the arginine pool of S. cerevisiae [12].

I I I I I

5 10 15 20 25 30 Time of incubation ( r n i n )

Fig.1. Kinetics of arginyl-tRNA synthesis as a function of enzyme concentration. Reaction mixtures as indicated in Materials and Methods. At given time intervals, aliquots were taken and precipitated with cold trichloroacetic acid. Symbols; (9) 1.25 pg protein (crude extract of wild-type Cells); ( X ) 2.50 pg; (A) 3.75 pg; (0) 5.00 pg

If arginyl-tRNA were the corepressor, the direct or indirect role of ornithine and citrulline on the regulation might be explained by their effect on the synthesis of arginyl-tRNA. I n view of the work of Ramos et al. [12], ornithine might activate arginyl- tRNA synthetase and thus increase the concentration of arginyl-tRNA, the eventual corepressor. Inversely, citrulline or one of its derivatives might inhibit arginyl-tRNA synthetase and lower the intracellular concentration of corepressor, and thus lead to a repression of ornithine transcarbamylase, not directly correlated to the intracellular arginine pool. We studied the effects on arginyl-tRNA synthetase of both these intermediates.

When measuring aminoacylation of tRNA as a function of arginine concentration, both in the presence and in the absence of ornithine or citrulline, no effect on the apparent affinity of arginine for the enzyme could be seen (Fig.2).

This absence of effect of ornithine and citrulline on aminoacylation in vitro agrees with the percent- ages of aminoacylation of tRNA*rg in vivo, which are not modified when cells are grown on these intermediates (see Table 1, experiments 3 and 4). The effects of ornithine and citrulline on the level of ornithine transcarbamylase (Table I, experiments 3 and 4) seem thus not to be correlated to modifications of tRNAArg aminoacylation in vitro or in vivo. These results are a t variance with the inhibitory effect of ornithine and citrulline on arginyl-tRNA synthetase, found in E. coli [17].


90 argR Mutations in Sacekromyces cerevisiae

'0-

1

Table 1. Percentage of aminoacylated tRNAArg in wild type, argR and argininosuccinase-lacking mutant strain of S. cerevisiae Activity of ornithine transcarbamylase is expressed as rate of citrulline formation

I

rq- XLP! g- ,i

---$-A--- I I

Expt Strains (genotype) Nitrogen source in culture mcdia Activity of ornithine tRNA*rg transcarbamylase aminoacylated

N O m g b l pnol x h-l O/O x mg protein-'

1 1278B (a ) (wild type) (NH,),SO, 1.2 35 68, 67, 67 2 (NH,),S04 + -4% 1.2 + 0.5 2 65, 68, 72 3 (NHd804 + 1.2 + 1 12 71, 75, 69 4 Citrulline 1.2 9 69, 69

62

71 -

0.2 1 0.2 + 0.2 1

8 MG542 (argH) A% 9 Arg + Citrulline

64 69

6o t

Fig. 2. Activity of nrginyl-tRNA synthetase for [Wlarginine in the presence or absence of products, affecting the regulation of arginine metabolism (plotted according to Lineweaver and Burle). Additions to reaction mixtures, containing crude cell-free extracts of wild-type cells: (-) no additions; (----) canavanine 0.4 mM final concentration; ( x ) citrnlline 6.6 mM; ( 0 ) homoarginine 6.6 mM; ( A ) ornithine 6.6 mM. Activity of enzyme is expressed as (counts/min)-' (see: Peculiarities of the aminoacylation reaction)

Homoarginine Homoarginine is an homologue of arginine ; when

added to the growth medium of a wild-type strain, homoarginine represses arginine biosynthetic enzymes. I n a regulatory mutant, argR, neither arginine nor homoarginine causes this repression anymore. Both arginine and homoarginine seem to take part in the same mechanism of repression, determined a t least in part by the argR gene, since by mutation of this gene, the mutated cells lose repressibility by both arginine and homoarginine. If the similarity of arginine and homoarginine in the repression was a t the level of their tRNA derivative, one excepts to find a competition for arginyl-tRNA synthetase between arginine and its homologue. Affinity of

arginyl-tRNA synthetase for arginine was measured both in the absence and in the presence of homoarginine in the reaction mixture. N o effect of homoarginine could be seen (see Fig.2). This was also done with a cell-free extract of argR mutant and no effect of homoarginine could be observed (not shown on the figure).

Argininosuccinic Acid In Neurospora crassa [9], Chlamydomonas [lo]

and E. coli [17], argininosuccinic acid, the immediate precursor of arginine, was shown to inhibit arginyl- tRNA synthetase. A mutant of Neurospora crmsa [9] lacking the last enzyme of the arginine biosynthetic pathway, argininosuccinase, had an increased intracellular pool of argininosuccinic acid, when grown on arginine, in the presence of citrulline. The level of ornithine transcarbamylase in the same conditions was derepressed and the percentage of arginyl-tRNA was reduced to 5O/, (instead of 39O/,, when citrulline was omitted from the growth medium). These studies suggested arginyl-tRNA to be the corepressor, instead of arginine itself, in the regulation of ornithine transcarbamylase.

The existence of such a mechanism in Xaccharo- rnyces cerevisiae was examined. The possible inhibitory effect of argininosuccinic acid on arginyl-tRNA synthetase was first examined.

Any contaminating arginine in the assay mixture would dilute the isotopically labelled arginine and interfere with the assay of arginyl-tRNA synthetase. Argininosuccinic acid was precipitated with alcohol before use to free the commercial preparation of any contaminating arginine. I n addition, care was taken to use as source of arginyl-tRNA synthetase an acellular extract of a mutant, lacking argininosuccinase. Fig. 3 shows Lineweaver-Burk plots of arginyl-tRNA synthetase activities as a function of arginine concentration, in the presence or absence of


P. P. Hoet and J.-M. Wiame 91

150 1 /= i

- 0 . 5 0 0.5 1 .o l / [“C-Arginine] (pM- ’ )

Fig. 3. Effect of argininosuccinate on aminoacylation of tRNAArp (plotted aceording to Lineweaver and Burk). (I) Arginino- succinic acid was precipitated with alcohol, centrifuged, resuspended in water and added to the reaction mixture (3.3 mM final concentration). Cell-free extract of mutant MG 542 was used, lacking argininosuccinase. (11) Arginino- succinic acid (3.3 mM), without previous alcoholic precipitation, was added to the reaction mixture. Cell-free extract of mutant MG 542, unable to convert argininosuccinate into arginine. (111) argininosuccinate (3.3 mM) previously precipitated with alcohol. Cell-free extract of wild-type cells. (IV) No argininosuccinate. Cell-free extract of mutant or wild-type cells. Activity of enzyme is expressed as (counts/ min)-l (see : Peculiarities of the aminoacylation reaction)

argininosuccinate. It shows the absence of inhibition of arginyl-tRNA synthetase by argininosuccinate, under the conditions described (Fig. 3, curve I). In addition it illustrates the two sources of isotopic dilution, to be avoided in this type of experiment; curve I1 has been obtained when using argininosuccinic acid, as such, without previous alcoholic precipitation. Curve I11 illustrates a second source of isotopic dilution. The conversion of the added argininosuccinic acid into arginine by an extract of wild-type cells (compare curve111 with curve I , where an extract was made from a mutant, lacking argininosuccinase). Conditions were realized in vivo in Saccharomyces cerevisiae similar to the ones described by Nazario in Neurospora crassa [9]. Mutant MG542 (argH-), lacking argininosuccinase was grown on minimal medium + 200 p g / d arginine, or on the same growth medium with, in addition, 200 pg/ml citrulline. Synthesis of ornithine transcarbamylase was repressed, irrespective of the presence or the absence of citrulline in the growth medium, and there was no deacylation of tRNAArg (Table I, expt 8-9). Derepression of ornithine transcarbamylase and discharging of tRNAArg could thus not be obtained in S. cerevisiae under conditions where they were observed in Neurospora.

The Percentage of tRNA*rg Charged in vivo and Its Relation to the Repression of Arginine Biosynthetic Enzymes

The percentage of charged tRNAArg was determined in an urgR mutant, grown in the presence or in the absence of arginine. As shown in TabIe 1 (Expt 5-7) even when arginine is present in the growth medium, ornithine transcarbamylase is not repressed, but this cannot be explained by a lack of arginyl-tRNA within the cells. argR mutations thus seem not to affect the synthesis of arginyl-tRNA, strengthening the hypothesis of mutational modzca- tions of a heteropolypeptidic aporepressor [13].

DISCUSSION

Ornithine, citrulline and homoarginine, having a regulatory role on arginine biosynthesis in the wild type but not in argR mutants, do not interfere with aminoacylation of tRNAArg. The same aminoacylation reaction does not seem to be modified in argR mutations, strengthening the hypothesis of a mutation, involving the aporepressor.

These conclusions result from the absence of effect of ornithine, citrulline, homoarginine and argininosuccinate on the aminoacylation reaction in vitro. These experiments are in agreement with the determinations of arginyl-tRNA in vivo.

No change in percentage of arginyl-tRNA could be observed in any of the cultures tested (see Table 1). I n particular, the absence of deacylation in argR mutants should be noted. Since the total quantity of tRNA extracted did not vary between the different cultures, the percentage of arginyl-tRNA is a measure of the total amount of arginyl-tRNA present in the cells.

Yet, one of the isoaccepting forms of arginyl- tRNA could have been specifically deacylated, without changing appreciably the percentage measured, if deacylation concerned one of the minor species. In a previous note [IS] we showed this to be unlikely, since no niodification appeared in the pattern obtained upon chromatographic separation of the isoaccepting species of arginyl-tRNA, extracted from wild type or argR mutant. The validity of this conclusion concerning a minor isoaccepting tRNA*rg rests on the sensitivity of the reversed phase chromatographic method used [I91 and the possibility of a small fraction of arginyl-tRNA being involved cannot be excluded, as discussed by Celis and Maas [20]. Moreover, one of the tRNAArg of yeast, behaving as an homogeneous peak on separation by three different chromatographic methods, was recently shown to be heterogeneous upon nucleotide sequence analysis [21]. Such tRNA heterogeneity might have an importance in regulation, and variation within this tRNAArg species would thus not be revealed by


92 P. P. Hoet and J.-M. Wiame: argR Mutations in Saccharomyces cerevisiae

reversed-phase chromatographic separation. The same observation would apply for modifications of tRNA, intervening after charging of tRNA with its homologous amino acid, as have been found important in the regulation of histidine biosynthesis in X. typhi- umrium [4].

With the help of an EMBO short term fellowship (to P. Hoet), the initial part of this work was started in the Laboratoire de Chimie Biologique, UniversitB Louis Pasteur, Strasbourg, under the guidance of Professors J. P. Ebel and J. H. Weil, to whom we express our sincere gratitude for their help. P. Hoet is Chercheur qualifie' du Fonds National de la Recherche scientifique, from Belgium.

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Avenue Chapelle-aux-Champs, 4,

J.-W. Wiame, Institut de Recherches du C.E.R.I.A., Avenue Emile-Gryzon, B-1070 Bruxelles, Belgium


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On the Nature of argR Mutations in Saccharomyces cerevisiae