502
Bwchimica at Biophysica Attn. 1073(1991)502-508 as 1991 ElsevierSciencePublishersB.V,0304-4165/91/$03.50 ADONIS 0304416591001258
BBAGEN 23479
Yeast proteinase yscB inactivates the leucyl tRNA synthetase in extracts of Saccharomyces cerevisiae I h i g o F. L a r r i n o a a n d C l a u d i a F. H e r e d i a In~tituta de Int.estiga¢iones Biornddictts de1 CS1C. Facuhad de Medicina de la UAM, Madrid (Spain)
(Received2g September 1990) K¢;)' wolds; Amitloacyl*tRNA5ylnh¢l~¢; Enz3/ra¢inarCtivalion;Proteolytieinae.tivaUon;Pr~teinase;S. ,'ereoi.~iae The aminoacyl-tRNA synthetases are inactivated in extracts of Saecharomyees cerecls~e preierentlany to other yeast ert~ymes and the rate of inactivation Keeatly increases in extracts of nilrogen-starved cells. The intensily of itmetiwfion varies for the different synehetases. Under conditions in which more than 80 per cent of the leucyl and i~leucyl.tRNA synthetases are inactivated, the activities of the synthetases for serine and arginine remain unchanged and the synthetases for other amino acids are inactivated to different extents. We have analyzed the characteristics ol inactivation of the leucyi-tRNA synthetase~ and identified the inactivating agent as the yeast proteinase yscB by the following eriterie: co-induction of both activities by nitrogen starvation; same Imlto'n of sensitivity to yeast proteinas¢ inhibitors; co-purification through a procedure desisned to purify the la'otelnase yseB and lack of inactivating activity in exlracts of a nitrogea-starYed yeast mutant lacking proteinase yscB.
Introduction The fundamental role played by the anfinoaeyl-tRNA synthetases in the decodification of the genetic message has prompted an intensive study of these enzymes both from structural and functional points of view, espeeiaUy in regards to their properties of specific recognition of transfer RNA and amino acids [1,2]. An obligatory step for these studies is the isolation and purification of these enzymes. Yeast has been widely used as a biological source for the purification of several synthetases. The handling of these enzymes is hindered by their apparent susceptibility to proteolytic attack [3], which renders different molecular forms differing in some properties. This sensitivity to proteinases could also be the cause of the low synthetase activities generally found in celt-free extracts from yeast compared with the intensity of the flux of protein biosynthesis in growing cells, 1t eouid also contribute to the lack of success in the isolation of syathetases complexes in yeast in contrast with that found in other eukaryotes [5,6] and the difficulty in obtaining cell-free extracts from yeast efficient
Correspondence:C,F. Hetedia,lnsti~utodc hlvcs~igaciofacsBiomEdicu,sd¢l CSIC, Facultadde Medicinad= ]a UAM, Arzobispo,Morcilla. 4, 2S029Madrid, Spain
to synthesize proteins [7]. The inactivation of the syn, thetases during the preparation of the cell-free extracts might also interfere with metabolic regulation studies of the synthesis of these enzymes by changes in cell growth conditions [8]. All of these reasons make it desirable to identify the proteinasc(s) responsible for synthetase inactivation. In this paper, we show that the yeast aminoacyl-tRNA synthetascs are inactivated in extracts of yeast preferentially to other yeast proteins. Our results indicate that the inactivation of the lencyl-tRNA synthetase is duc to the action of the yeast proteinase yscB.
Materials and Methods Materials. Uniformly labelled 14C-amino acids were obtained from the Amersham International {Amersham, U.K.). Crude brewers yeast tRNA was from Boehringer (Mannheim, F,R.G.). ATP, bovine serum albumin, az0coll and the proteinase inhibitors phenylmethylsalph0nyl fluoride (PMSF), soybean trypsin inhibitor (STI), N-zosyi L-phanylalanine ¢loromethyl ketone (TPCK) and Chymostatin A were from Sigma (St. Louis, MO, U.S.A.). Growth of the yeast and preparation o f the yeast extracts. The, strains of yeast used were Saccharomyces cerevisiae strain PM-1 [9] and the following mutants
5[)3 lacking the indicated proteinases: ABYS t (without proteinases A, B, Y, &); BYS (without proteinases B, "/, S); and Y H H 1 9 (without proteinase B). At] mutants were kindly supplied by Dr. Dieter H. 9 , o i l The nomenclature used for the proteinases is that previously used liD]. Yeasts were grown at 3 0 ° C with rotator3' shaking in a mineral medium [tl] with 2% glucc~.ge (w/v) as carbon and energy source. The medium was attjusted to p H 6 with potassium hydroxide. Conditions for ammonium and ~ucose starvation were those described elsewhere [12]. Preparation of yeast extracts. Cells (200 me, w / w ) were suspended m 1 ml of a solution containing 50 mM Tris-HC1 (pH 7), 10 rnM magnesium acetate, 10 mM 2-mercaptoethanol, 200 p g / m l soybean trypsin inhibitor (STI) and 0.2 mM PM~iF. The suspension was mixed with 2 g of glass beads (0.5 m m diameter) and vortexed four times for 1 rain periods, followed by inmersion for 1 rain intervals in an ice bath, The procedure can be scaled up maintaining the proportions indicated above. After vortexing, the suspension was centrifuged at low speed for 5 rain at 4~C to eliminate the glass beads a n d the supernatant was centrifuged at 100O0 × g for 30 rain. The supernatant was centrifuged at 105 000 x g for 90 rain and the supernatant of this centrifugation was used as source of the aminoacytt R N A synthetases. Analytical procedures. The activity of the aminoacyltRlqA synthetases was estimated as indicated ]13] in a reaction mixture (0.1 ml)containing 100 mM Hepes-HCI (pH 7.5); 10 mM magnesium acetate: 10 mM ATP (pH 7); 260/zg yeast tRNA; 10 mM 2-mereaptoethanol a n d the indicated ~aC-amino acid (0.1 raM) with a specific IO0
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Titan (h] Fig.. 2 Efrec! of a m m o n i u m a n d glucose starvation o n the aminoacyl-
iItNA s~mhetaseactivities ill the ye~t extracts. Exponentiallygrov,ins yeast in complete mineral medium were starv'~O,of the ~omponcnts described in Matg~mlsand Methods for the tim~ indicated. Contr.01~of extracts from non-slarvcd o=liswere gun in parallel. The activities".~eredetcrnfinedas in Fig. 1,
activity of 10 p,C/pmol. When indicated, a mixture of laC-amino acids (Radiochemical Center) was used at a final concentration of 14 pM for each amino acid. After incubation at 3 O ° C for up to 10 rain with the appropriate amount of the yeast extract, the reaction was slopped by the addition of 3 ml cold 5% trich[oroacetic acid (TCA). After 15 rain in an ice bath, the mixtures were filtered through glass fiber discs (Whatman GF), the filters washed three times with 3 ml of 5% cold TCA, dried and the radioactivity in the filters ~timated in a liquid scintillation spectrometer. Proteins were determined b y the method of Lowry et al. [14] using bovine serunt albumin as standard. Activities of the enzymes of the glycolytic pathway were determinod as described [15]. AU auxiliary enzymes were from l?.oehringer. The proteinase y s e g activity was measured as indi~ rated [16] by incubation at 3 0 ° C with vigorous stir ring, of 0.5 ml of a suspension of azocoil 2,5% in buffer 0.1 M potassiumphosphate(pH 7) with 0.1 ml of the yeast extract. The reaction was stopped b y addition of 0.5 rM of 10% TCA. After centrifugadon at 12000 rpm for 5 nfin, the increase iu absorbauc¢ at 520 run was estimated in the supernatant.
Results Aminoacy/-tRNA synthetases activities in extracts from yeasts growing in different conditions
J
Time
COnlrul t~r withGu! qlu¢@$e
Yeast g r o w t h in complete m e d i u m w a s
followed turbidimetrlcatly at 420 rim, Total ammoacyl-tRNA synth~titses activities were determined as described in Materials and Methods using the 14C-aminoacid mixture melnio~ed. 100%activity corresponds to 2.8 nmol amino acids incorporated per rng protein per mln.
The results in Fig. 1 show that the activities of the 0minoacyl-tRNA synthetascs in cell-free extracts greatly decrease when the yeast ceils enter the statlonm'y phase of growth. [n extracts from ceils in the late stationary phase, the ~ynthetase activities represent 40~ of those found in extracts from cells growing exponentially. To see the influence which the deprivation of the main components of the growth medium has on the syn-
504 thetase activities, growth medium was deprived of glucose and of the nitrogen source. The results in Fig. 2 show that nitrogen source depriva',ion results in a timedependent decay of the activities which reach valu,-~ of around 20% of the control in cell extracts which have been nitrogen starved for 5 h. This effect is not observed upon deprivation of glucose. The observed loss in synthetase activities could be due either to enzyme reactivation or to the presence of some synthetase inhibitor in extracts of the starved cells. To distinguish between these two possibilities, we performed znixture experiments with extracts from normal and rdtrogenstarved co|Is. The fact that the extracts from nitrogenstarved cells have no inhibitory effect on the synthetase activities present in extracts from cells growing in complete medium ruled out the presence of inhibitors and indicates a process of inactivation as the most probable mechanism. To derive information on the generality of this inactivation, we analyzed the behaviour of other enzymes in response to the deprivation of the yeast cells of their nitrogen source. The results of this experiment, presented in Fig. 3. show that the inaetlvation has some degree of specificity. It can be seen that, in contrast to the ease of the aminoacyl-tgNA synthetases (A), a number of enzymes of the glycolytic pathway (B) do not experiment siguificant variations upon deprivation of the nitrogen sourOe. Moreover, among the aminoacyltRNA syntheta~s, there is also certain degree of specificity (A). While seryl- and arginyl-tRNA synthetases are not affected at all, the activities of other synthetases decrease to values around 50% of the control, and other synthetases (leucyl and isoleucyl) are especially sensitive to inactivation. The degree of inactivation of the synthetases upon nitrogen starvation also depends on the nitrogen source used for yeast cell growth and is much more pronounced when ammonium sulfate is used rather than praline.
Characfemfics of the inactivation of the leucyl-tRNA aynthetase Once it was found that extracts from nitrogen-deprived yeast contain low Ievds of aminoacyl-tRNA synthetases and that leucyl-tRNA synthetase was one of the most affected, we centered our attention on characterizing the causes of the inactivation of this enzyme. In Table I it can be seen that, as shown above, nitrogen starvation results in a loss of enzymatic activity, while removal of the carbon source has no effect. However, when the carbon is removed together with the nitrogen source, the decrease of the leucyl-tRNA synthetas¢ activity which takes place by nitrogen deprivation does not occur. The same is true when eyeloheximide is added at the time of nitrogen starvation, indicating that protein biosynthesis is necessary for the inactivation of the synthetase. These results suggest that inactivation is
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Fig. 3. Diffe~tial effect of nitrogen stat~ati~n o n different enzymatic ~etivities, F~tponentially growing yeast in complete mineral media were s~rved of the nitrogen source for 5 h as desc~bed in Materials dud M¢~IIQds. Thc acti'dty of the synthetases (A) wa~ determined as described rain 8 each amino acid at a COncentration of l /tM except for glycine (4.5 /tM). The activities of the glycolytic enzymes (B) were determined as indicated in Malerials and Methods. Abbreviations: HK. hexokinas¢ (I/12 2.7.1.1.); PGt, 81ucoseFho~pl~ateismnerase (EC 5.3.1.931 G3PDH. 81yceraldehyde-3-pltosptmte dehydrogenase (EC 13.].9S.); ADH, alcohol dehydrogenate (EC I.I.I.L); GrPDH, glucose-6-phosphate dehydragena~ (F~ IA,L49.); I~K, pytuvate kinasc (EC 2.7.L40,), tO0g activity corresponds to the activities found in 1he cells jttst befo~ nitrogen starvalJon.
produced by ~ome enzyme which is synthesized in response to nitrogen starvation. It is well known that when yeast cells are deprived of nitrogen, the synthesis of several protelnascs is induced [10]. The possibility then existed that the observed inactivation of the aminoacyl-tRNA synthetase were the result of proteolyric degradation durilLg the preparation of the extracts, in spite of the fact that proteinase inhibitors were routinely included in all solutions at the concentrations recommended dsewher¢ [3]. The fact that increasing thc concentration o f PMSF in the solutions used for the preparation of the extracts from 0.2 to 5 mM produced no inactivation of the synthetase indicated that the inantivation was occurring during the preparation of the extracts and was probably due to the action of some proteinase.
503 TABLE l
Tyr Alp Pbe
l~[[ect of the gt~,~th condttzons on tke ~r:ut'ti~alron of the leucyt tRNA s~nthetase Expontmfiall~ growing yeast ce}ls were subJeCted for 5 h to the conditions indicated in the Table as described in Matetial~ and Method~. The leueyl-tRNA synthelase activity in the emmcts ',vas determined ~s des~l:~d in Materials and Methods. the inactivating enzyme as described in Fig. 4. and protei,"msey~B a~ indicated in Materials and Methods. 1CO%of tht |eueyl-tRNA ~yl~th¢t~e activity corresponds to 15 nmul of leucine incorpo,ato:l I~r m~ pr~te~n p~r min. Oxowlb. conditions
Leu¢~l.
tKNA synthetase activilv (%) Complete m~lium
Without ammonium (A) (A)+ eycloheximJde Without ammonium and 8htoose Without glucose Suaionary phase cells
Inactirating el~ynlC (arbitrary units)
Prot¢inas¢ ysctt (arbitrary units)
1 95
43
125
3 15
6
Prexen~e of the inactivming activily i~t exlrctet~ o[ nitrogen starved cells If the above hypothesis were correct, it should be possible m find the inactivating enzyme in extracts of nitrogen,starved ceils. T h e results in Fig. 4 show that, in fact, extracts from nitrogen-starved cells inactivate the leucyl-tRNA synthetas¢ present in extracts from cells grown in complete m e d i u m in a t i m ¢ - d q ~ n d e m reaction. The inactivation process is thermosensitive a n d is blocked by 5 m M PMSF, suggesting timt a proteolytic degradation is involved in the process, if this inactivab
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Fig. 5. Speckficity of the inactivatie ¢z~3qaz¢. One votum~ Qf exzract from yest grown in complel¢ medium wag mixed wi[h two ,~oIumes or extract item aitmgen-starve,J ceils. After 30 rain at 30°C. file remaining ac~i,dlie~ or the ePzymcs shown in the figure ~ e determined. Actlviti~ are expressed in per cent of t h ~ found at t~.me
4
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G3P~OH
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Ftg. 4. i~¢tiva~,/on of ~eug~I-LRNAsynzlmtas~ aclivi~ by extracL~of nitrogen-started cells. Extracts from yeast grown in complete medium were mixed and incubated at 300C with an equal volume of e~tracts [rora nitrogell-slarve~cells, either normal i U), heat-Ireated (o) (~0° C, S rain) orL~ tl~ p ~ e n ~ o f $ m M PMSF ( x ) . The re,dual lct~yltRNA synthetase activity wa~ determined at the indicated times in appt0priate aliquot~. Activity is expressed in ~tmolof leu¢ine incorporated l~X ml of (zcaction) mixtuLre.
ins e n z y m e is responsible for the observed inacdvations, its a p p e a r a n c e skould respoud to the same nutritional signah and its specificity characteristics should b¢ the s a m e as those shown in Fig. 3. In Table I it can be seen that the appearance o f the leucyl-tRNA-inaclivating e n z y m e parallels the inactivation of the synthetase. T h e inactivating e n z y m e appears only when the cells are deprived of the m t r o g e n source o r in stationary phase cells, a u d does not occur when glucose is also r e m o v e d or when eycloheximide is a d d e d to the yeast culture at the Lime of Ultrogon starvation. T h e specificity of inactivation by these extracts is shown in Fig. 5, which shows that it is qualitatively similar to titat in Fig. 3. T h e enzymes f r o m the glycolytic p a t h w a y are insensitive to the inactivating e n z y m e action, while the aminoaq/It R N A synthetases are inactivated, although with differoat effectiveness. L e u e y I - t R N A synthetase is the most efficiently inactivated, Thus, these results indicate that the inactivating e n z y m e is responsible for the observed inactivation of the lencyl-tRNA synthelase and probably also of the other a m i n o a c y l - t R N A syathelases. Identification o f the leucyl-tRNA inactivating activity w~th the yeast proteinase y s c B The inhibition of the 1oucyl-tRNA inactivating enzyme by proteinase inhibitors (Fig. 4) strongly suggests that this activity could be o n e of the yeast proteinases. Yeast contain four main proteinases which have been well characterized [10]: two endoproteinases yseA a n d yscIL and two catboxypeptidases yscY a n d yseS. T h e r e arc yeast mutants lacking these proteinases a n d those m u t a n t s have been of the u t m o s t importance to establish the involvement of these enzymes in metabolic processes [17]. T h e activity of the 5'east proteinases gToatly increase upon r e m o v a l of the nitrogen source
506 [10]. Tiffs is a common characteristic with leucyl-tRNA synthetase inactivating activity. To analyse the relationship between the inactivating enzyme and some of the yeast proteinases we tea.ted for the presence of the inactivating enzyme in extracts of several prot¢inaselacking yeast mutants (ABYS and BYS) which have been maintained under nitrogen starvation. Mutant ABYS lacks the four proteinases a n d mutant BYS lacks proteinase ysc B, and carboxypeptidases yscY and yseS but has proteinase ysc A. We could no detect leucyltRNA synthetase inactivating activity in extracts from these nitrogen-starved proteinase deficient strains (r¢snhs not shown) and consequently, the levels of leueylt R N A 5ynthetas¢ in these extracts were high in contrast to extracts from the wild-type. These results suggested that one of the yeast proteinases could be the inactivating enzyme, If this is the case, the leucyl-tRNA synthetase present in these mutant extracts should be inactivated by mixing with extracts of nitrogen-starved wild-type yeast which contains the four proteinases and high levels of inactivating activity (Fig. 4). We did not find inactivation of the synthetase (results not shown). This would indicate the presence of an inhibitor of the inactivating activity in the mutants extracts whleh did not allow Correlation of lack of proteinases activity in the mutants with lack of the inactivating enzyme. Consequently, we had to use other experimental approaches to ascertain the relationship between the prote4nases and the inactivating activity. Yeast proteinases have different pattern of sensitivity towards inhibitors [18] and they can be differentiated by these criteria. Of the four main yeast proteinases mentioned above, only the endopeptidase yscB and the carboxypeptidase yseY are inhibited b y PMSF. Since the inactivating enzyme is sensitive to PMSF (Fig. 4), it could be one of these two proteinases, Proteinase yseB is inhibited by chymostatin but not by zPCK.; the opposite is true for the earboxypeptidasc yseY. Fig, 6 shows that the inactivating activity present in extracts of the wild-type nitrogen starved cells is blocked by ¢hymostatin but is insensitive to TPCK, suggesting that proteinase yseB a n d the inactivating enzyme are the same entity. Another fact in support of this view is that both. activities respond similarly to variationS in growth conditions (Table 1) and co-purify through a fraetionalion procedure designed to purify the proteinase yscB (21) involving ammonium sulfate fractionation of the yeast eymsolic fraction followed by ehr~nta~ography on hydroxylapatite (Fig, 7). Finally, we have made use of a yeast mutant y H H 1 9 lacking proteinase yscB to confirm the relationship between this enzyme and the leucylt R N A synthetase inactivating activity. The results in Fig. 8 show that, in contrast to the almost complete absence of leueyl-tRNA synthetase acsivit2~"in the ease of "~hewild-type, extracts from the mutant strain starved of nitrogen have activity as high as that found in
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Fig, 6. Differentialsensitivityof the i~activatiagenzyme to ptoleinL~ inhibitors. One volume of extracts [roffl cells grown in complete medium was mixed with one volume of eAtra.~tfrom nitrogen-starved ¢¢115in the ~bsenoc(~) and in the pre~enceof chyrnos|atin 5/ali/ml (A) or in th~ pr~enc~ of TPCK 2 mM (o) as indicated, The residual I~ucyl-tRNA syntheta~ activity ilx the mixtures was detemfineM at time zero (e) and after incubation of the mixtures for 60 rain at 30o C. A~:tivityis expressedin Fmolor leucine incorporated per ml of nfixture. control cells grown in complete media. This suggests that extracts of the mutant devoid of proteioasc yscB a l s o lack the enzyme responsible for the inactivation of Z.5-
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Fig, "L H~droxyapalilecolumn chromatography of the leucyl-tRNA synthelase inactivating enzyme, A concentrated fra~tien (2.8 mJ.}of the enzyme prepared by disaolvi~ the ammonium salphale precipitate (90% sftturalion}of the yeast 1050OOxg superllatant fraction (Materials and Methyls) in the equilibration buffer, was applied to a column (0.8X 4 cm) or hydroxyapatite(Bin-Red) equilibrated with l0 mM petas~ium phasphale/]0 mM 2-mereapteelhaaol ~pH 7). The column was washed with 0.1 M potassium phosphate/t0 mM 2-mercaploe~anol (pH 7) anlil no absotbanee at 280 nm was detected and the ~lumn was then elated with a gradient 0A to 0,4 M of p~tassium phosphate(pH 7) containing 10 rr,M 2-mercaptoeth~tol. Fractions of 1 nil were 03l[ect~l and the aCliVitle~of the prot~a~,~c y[CUan44 the leueyl-tRNA inactivatin8 enzyme were c'~etelminedas indie~te~ in Materials and Melhnds and in the legend of Fi3, 4 (4119,Pm~einase y~cB;(×). ]~ucyl-tRNAsynth¢t~.~maclivafing.~¢tivity,Activitiesale expressedin arbitrary units 0, A2~.
507 IOO eo
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20
40 Time
60
[rain)
Fig. 8. Inactivationby proteinase yscB of Ihc Icucyl-tRNAsynthetase pres~l~t in exlracts from proteinase yscB deficient mulanls. Exlracls from strain YHHI9 starved of nitrogen source ,,rare incubated with (a) and without {o) a purified traction (Fig, 7) 121lof yeast p~oleinase yseB at 30"C. At the indicated times, the residual activity of the leueyl-tgNA s3'nthetasewas determined. 100%activitycorrespondato 4 nmol of letmine incorporated per mg of protein per rain. The activity in non statwc.dceils is 5 am01of leucine inre~otated pet' mg of protein per mia+
the leucyi-tRNA synthetase and consequently, that both activities are the same. In this ease, addition of a purified preparation of proteinasc yscB to these extracts should result in the inactivation of the leucyl-tRNA syntbetase. Fig. 8 demonstrates that incubation of the extracts alone does not result in inactivation of the syntbetase (in contrast to the case of wild-type), while addition of proteinase yscB to this deficient extract results in a time-dependem inactivation e l the synthetase.
Discussion
Although variations in the levels of the aminoaeylt R N A synthotases in response to nutritional changes in the growth media have been observed mainly in bacteria [81 they have also been observed in other cells including yeasts [19]. The correct estimation of the intensity of this phenomenon is frequently hindered by interfering processes which occur during the handling o[ cells or the preparation of the cell-free extracts. For example+ inactivation of these enzymes has been reported as one of the main causes of interference during the study of the derepression of a m i n o a e y l - t R N A syntherases synthesis by removal of amino acids from the growth media 120]. The results presented here show that the aminoacylt R N A syn;h.ta.~e ac~2~Sties greatly d~atlai~h i . ~xtracts from yeast in the stalionary phase of growth (Fig. 1) or from cells starved of their nitrogen source (Fig. 2). The
specificity of the inactivation process (Fig. 3) and the fact that all solutions used for the preparation of the extracts contained proteinase inhibitors at the concentrations recommended by other workers [3] would appear to rate out an indiscriminate extracelhilar proteolytic process as the cause of inactivation. However. a close examination of the problem demonstrated that yeast aminoacyl-tRNA synthelases are especially sensitive to inactivation during the preparation of the cell-free extracts as compared with other enzymatic activities (Fig_ 3)_ The specificity of this inactivation is observed even among the synthetases; while seryl and arginyltRNA synthetases are not affected, leucyl-tRNA syntbetase is the most sensitive to inactivation (Fig, 3). The rate of inactivation greatly increases in extracts from nitrogen-starved cells (Table 1). In nitrogen-starved cell extracts we have found an enzymatic activity which inactivates the leucyl-tRNA synthetase (Fig. 4). The specificity of these extracts to inactivate different yeast enzymes (Fig. 5) is qualitatively the same as that observed for synthetases inactivation (Fig. 3), and the appearance of the lencyl-tRNA synthetasc inactivating enzyme responds to the same nutritional signals as those which produce inactivation of the synthctases (Table 1). This inactivating activity shares a series of properties with the yeast proteinase yscB: its synthesis is induced by nitrogen starvation (Table 1); it has the same pattern of sensitivity to proteinases inhibitors (Fig+ 6) as proteinase yscB and differs with the other main yeast proteinases [18]. it co-purifies with yeast proteinase yscB in a protocol designed to purify the ptoteinase (Fig. 7) [21]. Finally, the inactivating enzyme is absent in extracts of a proteinase NscB deficient nitrogen-starved yeast mutant. These extracts contain normal levels of leocyl-tRNA synthetase which is inactivated upon addition of purified proteina~e B (Fig, 8). These properties identify the inactivating activity as yeast proteinase yscB, The aminoacyl-tRNA synthetases are enzymes with different Catalytic domains aligned in the polypeptide chain, starting from the amino terminal end [1], These active centers have been identified mainly by manipulation of the genes coding for several of these synthetases 11]. A complementary approach to this problem could be the dissection of the enzyme moleoule by proteolytic degradation, followed by analysis of specific functions associated with the resulting fragments, as it has been done with other enzymes, O u r results suggest that the inactivation of the leaeyl-tRNA synthetase involves proteolysis by the yeast proteinase yscl~ as the most likely mechanism although other more indirect and sophisticated mechanisms cannot be completely excluded. These findings could be of use not only to avoid the deleterious effect of the proteinase, but also for im potential use as a tool in structure and functional studies of the syntbetases.
508 Acknowledgements This work has been supported by a grant from ffondo Nacional para la Investigacibn Cientlfica PB87-0206. L F . L had a fcllowshlp from Gobleamo Vasco. We are gratefull to M. Asunci6n N a v a r r o for the typing o f the manuscript, and to Caty Mark and A. Coloma, for reading the manuscript.
Re|e~n~s ] Schimmel, P. (1987)Annu. Rev. Biochem. 56, 125-158. 2 Yams~ M. (1988) Celt 55, 739-741. 3 Kern, D.. Dietrich, A,, Fasiolo, Y., Renaud, M,, Giegv, R. and EbeL J.P. (1977) Biochimie59, 453-462. 4 ~irakoglu. B. and Waller, J.P. (1985.) Eur. J. Bir~hem. 149, 353-q6]. 5 Dahg, Ch.V. and Dang. Ch.V. (1986) Biocher~. J. 239, 249-255. 6 Det*tcher, M.P. (1984) J, Cell. Biol. 9% 375-377. 7 Hofbauer, R.. Fe~sl, F., Hamilton, B. and Ruis. H, (1982) Eur. J. Biochem. 122, 19~203.
g Neidhardt, F.C.. Parker. J. and Mekoover, W.G, (1975) Annu. P,.ev. Mic.~ohiol.29, 215-250, 9 Hetedia, C.F., Dc la Fuenm, G. and :~lls, A. (19fi4) Biochim. Biophys. Acta 86. 216-223. 10 Achstetler, l". and Wolf, D,H. (1985) Yeast I, 139-157. 11 Lagunas, R, (1976) Biochim. Biophys. Acla 440, 651-674, 12 Lagunas, R_ (1979) MoL Cell. Biochem, 27, 139-146. 13 Del Rio, J.M. and Heredia, CF. (1993) Mol. Cell. Bioehem. 50. 101-106, 14 Lowry, H.O., Ro~eb,o~tg=h, N.J., Faxr, A.L. and Randall, R..[. (1951) J. Biol. Chem. 193, 265-275. 15 Betgmeyer, H.U. (1983) Methods of Enzymatic Analysis. (Bergmeyer, H.U., B~rgmeyer, J, and Graby, M., eds.). Veflag Chemle. 3Td Edn. Weinheim. 16 $ahekL T. and Holzer, H. (I975) Biochim. Biophys. Ac~ 38~.. 203-214. 17 Wolf, D.H. (1982) Tl~tds. Biol. Sci. 7, 35-37. 18 Wolf, D.H. (1986) Mi¢robiol. Sci. 3,107-114. 19 Johnson, R.C.. Vaaalta. PR. and Fresco. J.R. (1977) J. Biol. Chem. 252, 878-882. 20 Williams, L.S. and N=idhardt, F.C. (1969) J. Mol. Biol, 43, 52921 Katsunuma, T., ,%hott, E., EIssaser, S. and Holzer, H. (1972) Eur. J. giochem, 27, 520-526.
Bwchimica at Biophysica Attn. 1073(1991)502-508 as 1991 ElsevierSciencePublishersB.V,0304-4165/91/$03.50 ADONIS 0304416591001258
BBAGEN 23479
Yeast proteinase yscB inactivates the leucyl tRNA synthetase in extracts of Saccharomyces cerevisiae I h i g o F. L a r r i n o a a n d C l a u d i a F. H e r e d i a In~tituta de Int.estiga¢iones Biornddictts de1 CS1C. Facuhad de Medicina de la UAM, Madrid (Spain)
(Received2g September 1990) K¢;)' wolds; Amitloacyl*tRNA5ylnh¢l~¢; Enz3/ra¢inarCtivalion;Proteolytieinae.tivaUon;Pr~teinase;S. ,'ereoi.~iae The aminoacyl-tRNA synthetases are inactivated in extracts of Saecharomyees cerecls~e preierentlany to other yeast ert~ymes and the rate of inactivation Keeatly increases in extracts of nilrogen-starved cells. The intensily of itmetiwfion varies for the different synehetases. Under conditions in which more than 80 per cent of the leucyl and i~leucyl.tRNA synthetases are inactivated, the activities of the synthetases for serine and arginine remain unchanged and the synthetases for other amino acids are inactivated to different extents. We have analyzed the characteristics ol inactivation of the leucyi-tRNA synthetase~ and identified the inactivating agent as the yeast proteinase yscB by the following eriterie: co-induction of both activities by nitrogen starvation; same Imlto'n of sensitivity to yeast proteinas¢ inhibitors; co-purification through a procedure desisned to purify the la'otelnase yseB and lack of inactivating activity in exlracts of a nitrogea-starYed yeast mutant lacking proteinase yscB.
Introduction The fundamental role played by the anfinoaeyl-tRNA synthetases in the decodification of the genetic message has prompted an intensive study of these enzymes both from structural and functional points of view, espeeiaUy in regards to their properties of specific recognition of transfer RNA and amino acids [1,2]. An obligatory step for these studies is the isolation and purification of these enzymes. Yeast has been widely used as a biological source for the purification of several synthetases. The handling of these enzymes is hindered by their apparent susceptibility to proteolytic attack [3], which renders different molecular forms differing in some properties. This sensitivity to proteinases could also be the cause of the low synthetase activities generally found in celt-free extracts from yeast compared with the intensity of the flux of protein biosynthesis in growing cells, 1t eouid also contribute to the lack of success in the isolation of syathetases complexes in yeast in contrast with that found in other eukaryotes [5,6] and the difficulty in obtaining cell-free extracts from yeast efficient
Correspondence:C,F. Hetedia,lnsti~utodc hlvcs~igaciofacsBiomEdicu,sd¢l CSIC, Facultadde Medicinad= ]a UAM, Arzobispo,Morcilla. 4, 2S029Madrid, Spain
to synthesize proteins [7]. The inactivation of the syn, thetases during the preparation of the cell-free extracts might also interfere with metabolic regulation studies of the synthesis of these enzymes by changes in cell growth conditions [8]. All of these reasons make it desirable to identify the proteinasc(s) responsible for synthetase inactivation. In this paper, we show that the yeast aminoacyl-tRNA synthetascs are inactivated in extracts of yeast preferentially to other yeast proteins. Our results indicate that the inactivation of the lencyl-tRNA synthetase is duc to the action of the yeast proteinase yscB.
Materials and Methods Materials. Uniformly labelled 14C-amino acids were obtained from the Amersham International {Amersham, U.K.). Crude brewers yeast tRNA was from Boehringer (Mannheim, F,R.G.). ATP, bovine serum albumin, az0coll and the proteinase inhibitors phenylmethylsalph0nyl fluoride (PMSF), soybean trypsin inhibitor (STI), N-zosyi L-phanylalanine ¢loromethyl ketone (TPCK) and Chymostatin A were from Sigma (St. Louis, MO, U.S.A.). Growth of the yeast and preparation o f the yeast extracts. The, strains of yeast used were Saccharomyces cerevisiae strain PM-1 [9] and the following mutants
5[)3 lacking the indicated proteinases: ABYS t (without proteinases A, B, Y, &); BYS (without proteinases B, "/, S); and Y H H 1 9 (without proteinase B). At] mutants were kindly supplied by Dr. Dieter H. 9 , o i l The nomenclature used for the proteinases is that previously used liD]. Yeasts were grown at 3 0 ° C with rotator3' shaking in a mineral medium [tl] with 2% glucc~.ge (w/v) as carbon and energy source. The medium was attjusted to p H 6 with potassium hydroxide. Conditions for ammonium and ~ucose starvation were those described elsewhere [12]. Preparation of yeast extracts. Cells (200 me, w / w ) were suspended m 1 ml of a solution containing 50 mM Tris-HC1 (pH 7), 10 rnM magnesium acetate, 10 mM 2-mercaptoethanol, 200 p g / m l soybean trypsin inhibitor (STI) and 0.2 mM PM~iF. The suspension was mixed with 2 g of glass beads (0.5 m m diameter) and vortexed four times for 1 rain periods, followed by inmersion for 1 rain intervals in an ice bath, The procedure can be scaled up maintaining the proportions indicated above. After vortexing, the suspension was centrifuged at low speed for 5 rain at 4~C to eliminate the glass beads a n d the supernatant was centrifuged at 100O0 × g for 30 rain. The supernatant was centrifuged at 105 000 x g for 90 rain and the supernatant of this centrifugation was used as source of the aminoacytt R N A synthetases. Analytical procedures. The activity of the aminoacyltRlqA synthetases was estimated as indicated ]13] in a reaction mixture (0.1 ml)containing 100 mM Hepes-HCI (pH 7.5); 10 mM magnesium acetate: 10 mM ATP (pH 7); 260/zg yeast tRNA; 10 mM 2-mereaptoethanol a n d the indicated ~aC-amino acid (0.1 raM) with a specific IO0
E g 11"
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oo Z
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Fig. L Aminoacyl-tRNA synthcta~ in cxtxacts of yeasts 8rowing in complete mineral ~ i ~ L
~
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o
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,~
zo J L
I
t
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2
4
6
Titan (h] Fig.. 2 Efrec! of a m m o n i u m a n d glucose starvation o n the aminoacyl-
iItNA s~mhetaseactivities ill the ye~t extracts. Exponentiallygrov,ins yeast in complete mineral medium were starv'~O,of the ~omponcnts described in Matg~mlsand Methods for the tim~ indicated. Contr.01~of extracts from non-slarvcd o=liswere gun in parallel. The activities".~eredetcrnfinedas in Fig. 1,
activity of 10 p,C/pmol. When indicated, a mixture of laC-amino acids (Radiochemical Center) was used at a final concentration of 14 pM for each amino acid. After incubation at 3 O ° C for up to 10 rain with the appropriate amount of the yeast extract, the reaction was slopped by the addition of 3 ml cold 5% trich[oroacetic acid (TCA). After 15 rain in an ice bath, the mixtures were filtered through glass fiber discs (Whatman GF), the filters washed three times with 3 ml of 5% cold TCA, dried and the radioactivity in the filters ~timated in a liquid scintillation spectrometer. Proteins were determined b y the method of Lowry et al. [14] using bovine serunt albumin as standard. Activities of the enzymes of the glycolytic pathway were determinod as described [15]. AU auxiliary enzymes were from l?.oehringer. The proteinase y s e g activity was measured as indi~ rated [16] by incubation at 3 0 ° C with vigorous stir ring, of 0.5 ml of a suspension of azocoil 2,5% in buffer 0.1 M potassiumphosphate(pH 7) with 0.1 ml of the yeast extract. The reaction was stopped b y addition of 0.5 rM of 10% TCA. After centrifugadon at 12000 rpm for 5 nfin, the increase iu absorbauc¢ at 520 run was estimated in the supernatant.
Results Aminoacy/-tRNA synthetases activities in extracts from yeasts growing in different conditions
J
Time
COnlrul t~r withGu! qlu¢@$e
Yeast g r o w t h in complete m e d i u m w a s
followed turbidimetrlcatly at 420 rim, Total ammoacyl-tRNA synth~titses activities were determined as described in Materials and Methods using the 14C-aminoacid mixture melnio~ed. 100%activity corresponds to 2.8 nmol amino acids incorporated per rng protein per mln.
The results in Fig. 1 show that the activities of the 0minoacyl-tRNA synthetascs in cell-free extracts greatly decrease when the yeast ceils enter the statlonm'y phase of growth. [n extracts from ceils in the late stationary phase, the ~ynthetase activities represent 40~ of those found in extracts from cells growing exponentially. To see the influence which the deprivation of the main components of the growth medium has on the syn-
504 thetase activities, growth medium was deprived of glucose and of the nitrogen source. The results in Fig. 2 show that nitrogen source depriva',ion results in a timedependent decay of the activities which reach valu,-~ of around 20% of the control in cell extracts which have been nitrogen starved for 5 h. This effect is not observed upon deprivation of glucose. The observed loss in synthetase activities could be due either to enzyme reactivation or to the presence of some synthetase inhibitor in extracts of the starved cells. To distinguish between these two possibilities, we performed znixture experiments with extracts from normal and rdtrogenstarved co|Is. The fact that the extracts from nitrogenstarved cells have no inhibitory effect on the synthetase activities present in extracts from cells growing in complete medium ruled out the presence of inhibitors and indicates a process of inactivation as the most probable mechanism. To derive information on the generality of this inactivation, we analyzed the behaviour of other enzymes in response to the deprivation of the yeast cells of their nitrogen source. The results of this experiment, presented in Fig. 3. show that the inaetlvation has some degree of specificity. It can be seen that, in contrast to the ease of the aminoacyl-tgNA synthetases (A), a number of enzymes of the glycolytic pathway (B) do not experiment siguificant variations upon deprivation of the nitrogen sourOe. Moreover, among the aminoacyltRNA syntheta~s, there is also certain degree of specificity (A). While seryl- and arginyl-tRNA synthetases are not affected at all, the activities of other synthetases decrease to values around 50% of the control, and other synthetases (leucyl and isoleucyl) are especially sensitive to inactivation. The degree of inactivation of the synthetases upon nitrogen starvation also depends on the nitrogen source used for yeast cell growth and is much more pronounced when ammonium sulfate is used rather than praline.
Characfemfics of the inactivation of the leucyl-tRNA aynthetase Once it was found that extracts from nitrogen-deprived yeast contain low Ievds of aminoacyl-tRNA synthetases and that leucyl-tRNA synthetase was one of the most affected, we centered our attention on characterizing the causes of the inactivation of this enzyme. In Table I it can be seen that, as shown above, nitrogen starvation results in a loss of enzymatic activity, while removal of the carbon source has no effect. However, when the carbon is removed together with the nitrogen source, the decrease of the leucyl-tRNA synthetas¢ activity which takes place by nitrogen deprivation does not occur. The same is true when eyeloheximide is added at the time of nitrogen starvation, indicating that protein biosynthesis is necessary for the inactivation of the synthetase. These results suggest that inactivation is
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Fig. 3. Diffe~tial effect of nitrogen stat~ati~n o n different enzymatic ~etivities, F~tponentially growing yeast in complete mineral media were s~rved of the nitrogen source for 5 h as desc~bed in Materials dud M¢~IIQds. Thc acti'dty of the synthetases (A) wa~ determined as described rain 8 each amino acid at a COncentration of l /tM except for glycine (4.5 /tM). The activities of the glycolytic enzymes (B) were determined as indicated in Malerials and Methods. Abbreviations: HK. hexokinas¢ (I/12 2.7.1.1.); PGt, 81ucoseFho~pl~ateismnerase (EC 5.3.1.931 G3PDH. 81yceraldehyde-3-pltosptmte dehydrogenase (EC 13.].9S.); ADH, alcohol dehydrogenate (EC I.I.I.L); GrPDH, glucose-6-phosphate dehydragena~ (F~ IA,L49.); I~K, pytuvate kinasc (EC 2.7.L40,), tO0g activity corresponds to the activities found in 1he cells jttst befo~ nitrogen starvalJon.
produced by ~ome enzyme which is synthesized in response to nitrogen starvation. It is well known that when yeast cells are deprived of nitrogen, the synthesis of several protelnascs is induced [10]. The possibility then existed that the observed inactivation of the aminoacyl-tRNA synthetase were the result of proteolyric degradation durilLg the preparation of the extracts, in spite of the fact that proteinase inhibitors were routinely included in all solutions at the concentrations recommended dsewher¢ [3]. The fact that increasing thc concentration o f PMSF in the solutions used for the preparation of the extracts from 0.2 to 5 mM produced no inactivation of the synthetase indicated that the inantivation was occurring during the preparation of the extracts and was probably due to the action of some proteinase.
503 TABLE l
Tyr Alp Pbe
l~[[ect of the gt~,~th condttzons on tke ~r:ut'ti~alron of the leucyt tRNA s~nthetase Expontmfiall~ growing yeast ce}ls were subJeCted for 5 h to the conditions indicated in the Table as described in Matetial~ and Method~. The leueyl-tRNA synthelase activity in the emmcts ',vas determined ~s des~l:~d in Materials and Methods. the inactivating enzyme as described in Fig. 4. and protei,"msey~B a~ indicated in Materials and Methods. 1CO%of tht |eueyl-tRNA ~yl~th¢t~e activity corresponds to 15 nmul of leucine incorpo,ato:l I~r m~ pr~te~n p~r min. Oxowlb. conditions
Leu¢~l.
tKNA synthetase activilv (%) Complete m~lium
Without ammonium (A) (A)+ eycloheximJde Without ammonium and 8htoose Without glucose Suaionary phase cells
Inactirating el~ynlC (arbitrary units)
Prot¢inas¢ ysctt (arbitrary units)
1 95
43
125
3 15
6
Prexen~e of the inactivming activily i~t exlrctet~ o[ nitrogen starved cells If the above hypothesis were correct, it should be possible m find the inactivating enzyme in extracts of nitrogen,starved ceils. T h e results in Fig. 4 show that, in fact, extracts from nitrogen-starved cells inactivate the leucyl-tRNA synthetas¢ present in extracts from cells grown in complete m e d i u m in a t i m ¢ - d q ~ n d e m reaction. The inactivation process is thermosensitive a n d is blocked by 5 m M PMSF, suggesting timt a proteolytic degradation is involved in the process, if this inactivab
i
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i
20
40
60
Time
HK Gly Ly$ V~r Ale
T~
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2o
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n6 97 71
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Fig. 5. Speckficity of the inactivatie ¢z~3qaz¢. One votum~ Qf exzract from yest grown in complel¢ medium wag mixed wi[h two ,~oIumes or extract item aitmgen-starve,J ceils. After 30 rain at 30°C. file remaining ac~i,dlie~ or the ePzymcs shown in the figure ~ e determined. Actlviti~ are expressed in per cent of t h ~ found at t~.me
4
100
G3P~OH
I00
(raM)
Ftg. 4. i~¢tiva~,/on of ~eug~I-LRNAsynzlmtas~ aclivi~ by extracL~of nitrogen-started cells. Extracts from yeast grown in complete medium were mixed and incubated at 300C with an equal volume of e~tracts [rora nitrogell-slarve~cells, either normal i U), heat-Ireated (o) (~0° C, S rain) orL~ tl~ p ~ e n ~ o f $ m M PMSF ( x ) . The re,dual lct~yltRNA synthetase activity wa~ determined at the indicated times in appt0priate aliquot~. Activity is expressed in ~tmolof leu¢ine incorporated l~X ml of (zcaction) mixtuLre.
ins e n z y m e is responsible for the observed inacdvations, its a p p e a r a n c e skould respoud to the same nutritional signah and its specificity characteristics should b¢ the s a m e as those shown in Fig. 3. In Table I it can be seen that the appearance o f the leucyl-tRNA-inaclivating e n z y m e parallels the inactivation of the synthetase. T h e inactivating e n z y m e appears only when the cells are deprived of the m t r o g e n source o r in stationary phase cells, a u d does not occur when glucose is also r e m o v e d or when eycloheximide is a d d e d to the yeast culture at the Lime of Ultrogon starvation. T h e specificity of inactivation by these extracts is shown in Fig. 5, which shows that it is qualitatively similar to titat in Fig. 3. T h e enzymes f r o m the glycolytic p a t h w a y are insensitive to the inactivating e n z y m e action, while the aminoaq/It R N A synthetases are inactivated, although with differoat effectiveness. L e u e y I - t R N A synthetase is the most efficiently inactivated, Thus, these results indicate that the inactivating e n z y m e is responsible for the observed inactivation of the lencyl-tRNA synthelase and probably also of the other a m i n o a c y l - t R N A syathelases. Identification o f the leucyl-tRNA inactivating activity w~th the yeast proteinase y s c B The inhibition of the 1oucyl-tRNA inactivating enzyme by proteinase inhibitors (Fig. 4) strongly suggests that this activity could be o n e of the yeast proteinases. Yeast contain four main proteinases which have been well characterized [10]: two endoproteinases yseA a n d yscIL and two catboxypeptidases yscY a n d yseS. T h e r e arc yeast mutants lacking these proteinases a n d those m u t a n t s have been of the u t m o s t importance to establish the involvement of these enzymes in metabolic processes [17]. T h e activity of the 5'east proteinases gToatly increase upon r e m o v a l of the nitrogen source
506 [10]. Tiffs is a common characteristic with leucyl-tRNA synthetase inactivating activity. To analyse the relationship between the inactivating enzyme and some of the yeast proteinases we tea.ted for the presence of the inactivating enzyme in extracts of several prot¢inaselacking yeast mutants (ABYS and BYS) which have been maintained under nitrogen starvation. Mutant ABYS lacks the four proteinases a n d mutant BYS lacks proteinase ysc B, and carboxypeptidases yscY and yseS but has proteinase ysc A. We could no detect leucyltRNA synthetase inactivating activity in extracts from these nitrogen-starved proteinase deficient strains (r¢snhs not shown) and consequently, the levels of leueylt R N A 5ynthetas¢ in these extracts were high in contrast to extracts from the wild-type. These results suggested that one of the yeast proteinases could be the inactivating enzyme, If this is the case, the leucyl-tRNA synthetase present in these mutant extracts should be inactivated by mixing with extracts of nitrogen-starved wild-type yeast which contains the four proteinases and high levels of inactivating activity (Fig. 4). We did not find inactivation of the synthetase (results not shown). This would indicate the presence of an inhibitor of the inactivating activity in the mutants extracts whleh did not allow Correlation of lack of proteinases activity in the mutants with lack of the inactivating enzyme. Consequently, we had to use other experimental approaches to ascertain the relationship between the prote4nases and the inactivating activity. Yeast proteinases have different pattern of sensitivity towards inhibitors [18] and they can be differentiated by these criteria. Of the four main yeast proteinases mentioned above, only the endopeptidase yscB and the carboxypeptidase yseY are inhibited b y PMSF. Since the inactivating enzyme is sensitive to PMSF (Fig. 4), it could be one of these two proteinases, Proteinase yseB is inhibited by chymostatin but not by zPCK.; the opposite is true for the earboxypeptidasc yseY. Fig, 6 shows that the inactivating activity present in extracts of the wild-type nitrogen starved cells is blocked by ¢hymostatin but is insensitive to TPCK, suggesting that proteinase yseB a n d the inactivating enzyme are the same entity. Another fact in support of this view is that both. activities respond similarly to variationS in growth conditions (Table 1) and co-purify through a fraetionalion procedure designed to purify the proteinase yscB (21) involving ammonium sulfate fractionation of the yeast eymsolic fraction followed by ehr~nta~ography on hydroxylapatite (Fig, 7). Finally, we have made use of a yeast mutant y H H 1 9 lacking proteinase yscB to confirm the relationship between this enzyme and the leucylt R N A synthetase inactivating activity. The results in Fig. 8 show that, in contrast to the almost complete absence of leueyl-tRNA synthetase acsivit2~"in the ease of "~hewild-type, extracts from the mutant strain starved of nitrogen have activity as high as that found in
'.01 .//
i °t -
o,.j
A
0.4
0.~
Time
(rain)
Fig, 6. Differentialsensitivityof the i~activatiagenzyme to ptoleinL~ inhibitors. One volume of extracts [roffl cells grown in complete medium was mixed with one volume of eAtra.~tfrom nitrogen-starved ¢¢115in the ~bsenoc(~) and in the pre~enceof chyrnos|atin 5/ali/ml (A) or in th~ pr~enc~ of TPCK 2 mM (o) as indicated, The residual I~ucyl-tRNA syntheta~ activity ilx the mixtures was detemfineM at time zero (e) and after incubation of the mixtures for 60 rain at 30o C. A~:tivityis expressedin Fmolor leucine incorporated per ml of nfixture. control cells grown in complete media. This suggests that extracts of the mutant devoid of proteioasc yscB a l s o lack the enzyme responsible for the inactivation of Z.5-
/
0.4
x
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Fig, "L H~droxyapalilecolumn chromatography of the leucyl-tRNA synthelase inactivating enzyme, A concentrated fra~tien (2.8 mJ.}of the enzyme prepared by disaolvi~ the ammonium salphale precipitate (90% sftturalion}of the yeast 1050OOxg superllatant fraction (Materials and Methyls) in the equilibration buffer, was applied to a column (0.8X 4 cm) or hydroxyapatite(Bin-Red) equilibrated with l0 mM petas~ium phasphale/]0 mM 2-mereapteelhaaol ~pH 7). The column was washed with 0.1 M potassium phosphate/t0 mM 2-mercaploe~anol (pH 7) anlil no absotbanee at 280 nm was detected and the ~lumn was then elated with a gradient 0A to 0,4 M of p~tassium phosphate(pH 7) containing 10 rr,M 2-mercaptoeth~tol. Fractions of 1 nil were 03l[ect~l and the aCliVitle~of the prot~a~,~c y[CUan44 the leueyl-tRNA inactivatin8 enzyme were c'~etelminedas indie~te~ in Materials and Melhnds and in the legend of Fi3, 4 (4119,Pm~einase y~cB;(×). ]~ucyl-tRNAsynth¢t~.~maclivafing.~¢tivity,Activitiesale expressedin arbitrary units 0, A2~.
507 IOO eo
~
.
o c0n;rol
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i +° go 2o
20
40 Time
60
[rain)
Fig. 8. Inactivationby proteinase yscB of Ihc Icucyl-tRNAsynthetase pres~l~t in exlracts from proteinase yscB deficient mulanls. Exlracls from strain YHHI9 starved of nitrogen source ,,rare incubated with (a) and without {o) a purified traction (Fig, 7) 121lof yeast p~oleinase yseB at 30"C. At the indicated times, the residual activity of the leueyl-tgNA s3'nthetasewas determined. 100%activitycorrespondato 4 nmol of letmine incorporated per mg of protein per rain. The activity in non statwc.dceils is 5 am01of leucine inre~otated pet' mg of protein per mia+
the leucyi-tRNA synthetase and consequently, that both activities are the same. In this ease, addition of a purified preparation of proteinasc yscB to these extracts should result in the inactivation of the leucyl-tRNA syntbetase. Fig. 8 demonstrates that incubation of the extracts alone does not result in inactivation of the syntbetase (in contrast to the case of wild-type), while addition of proteinase yscB to this deficient extract results in a time-dependem inactivation e l the synthetase.
Discussion
Although variations in the levels of the aminoaeylt R N A synthotases in response to nutritional changes in the growth media have been observed mainly in bacteria [81 they have also been observed in other cells including yeasts [19]. The correct estimation of the intensity of this phenomenon is frequently hindered by interfering processes which occur during the handling o[ cells or the preparation of the cell-free extracts. For example+ inactivation of these enzymes has been reported as one of the main causes of interference during the study of the derepression of a m i n o a e y l - t R N A syntherases synthesis by removal of amino acids from the growth media 120]. The results presented here show that the aminoacylt R N A syn;h.ta.~e ac~2~Sties greatly d~atlai~h i . ~xtracts from yeast in the stalionary phase of growth (Fig. 1) or from cells starved of their nitrogen source (Fig. 2). The
specificity of the inactivation process (Fig. 3) and the fact that all solutions used for the preparation of the extracts contained proteinase inhibitors at the concentrations recommended by other workers [3] would appear to rate out an indiscriminate extracelhilar proteolytic process as the cause of inactivation. However. a close examination of the problem demonstrated that yeast aminoacyl-tRNA synthelases are especially sensitive to inactivation during the preparation of the cell-free extracts as compared with other enzymatic activities (Fig_ 3)_ The specificity of this inactivation is observed even among the synthetases; while seryl and arginyltRNA synthetases are not affected, leucyl-tRNA syntbetase is the most sensitive to inactivation (Fig, 3). The rate of inactivation greatly increases in extracts from nitrogen-starved cells (Table 1). In nitrogen-starved cell extracts we have found an enzymatic activity which inactivates the leucyl-tRNA synthetase (Fig. 4). The specificity of these extracts to inactivate different yeast enzymes (Fig. 5) is qualitatively the same as that observed for synthetases inactivation (Fig. 3), and the appearance of the lencyl-tRNA synthetasc inactivating enzyme responds to the same nutritional signals as those which produce inactivation of the synthctases (Table 1). This inactivating activity shares a series of properties with the yeast proteinase yscB: its synthesis is induced by nitrogen starvation (Table 1); it has the same pattern of sensitivity to proteinases inhibitors (Fig+ 6) as proteinase yscB and differs with the other main yeast proteinases [18]. it co-purifies with yeast proteinase yscB in a protocol designed to purify the ptoteinase (Fig. 7) [21]. Finally, the inactivating enzyme is absent in extracts of a proteinase NscB deficient nitrogen-starved yeast mutant. These extracts contain normal levels of leocyl-tRNA synthetase which is inactivated upon addition of purified proteina~e B (Fig, 8). These properties identify the inactivating activity as yeast proteinase yscB, The aminoacyl-tRNA synthetases are enzymes with different Catalytic domains aligned in the polypeptide chain, starting from the amino terminal end [1], These active centers have been identified mainly by manipulation of the genes coding for several of these synthetases 11]. A complementary approach to this problem could be the dissection of the enzyme moleoule by proteolytic degradation, followed by analysis of specific functions associated with the resulting fragments, as it has been done with other enzymes, O u r results suggest that the inactivation of the leaeyl-tRNA synthetase involves proteolysis by the yeast proteinase yscl~ as the most likely mechanism although other more indirect and sophisticated mechanisms cannot be completely excluded. These findings could be of use not only to avoid the deleterious effect of the proteinase, but also for im potential use as a tool in structure and functional studies of the syntbetases.
508 Acknowledgements This work has been supported by a grant from ffondo Nacional para la Investigacibn Cientlfica PB87-0206. L F . L had a fcllowshlp from Gobleamo Vasco. We are gratefull to M. Asunci6n N a v a r r o for the typing o f the manuscript, and to Caty Mark and A. Coloma, for reading the manuscript.
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