Aminoacylation of Phaseolus vulgaris cytoplasmic, chloroplastic and mitochondrial tRNAsMet and of Escherichia coli tRNAsMet by homologous and heterologous enzymes

240

Biochimica et Biophysica A cta, 407 (1975) 240--248

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98416 AMINOACYLATION OF P H A S E O L U S V U L G A R I S CYTOPLASMIC, CHLOROPLASTIC AND MITOCHONDRIAL tRNAs Met AND OF E S C H E R I C H I A C O L I tRNAs Met BY HOMOLOGOUS AND HETEROLOGOUS ENZYMES

P. GUILLEMAUT and J.H. WEIL Institut de Biologie Mol~culaire et Cellulaire, Universitd Louis Pasteur, 15 rue Descartes, Esplanade, 67000 Strasbourg (France)

(Received May 21st, 1975)

Summary Met-tRNA synthetase from Phaseolus vulgaris cytoplasm could be separated from its chloroplastic or mitochondrial counterpart by DEAE-cellulose chromatography, but the Met-tRNA synthetase from the two latter organelles could n o t be distinguished using DEAE-cellulose, h y d r o x y a p a t i t e or CM-Sephadex chromatography. As revealed by reverse-phase chromatography, bean cytoplasm contains 2 tRNAs Met; only one is charged by chloroplast, mitochondrial or Escherichia coli Met-tRNA synthetase. Mitochondria contain, in addition to the 2 cytoplasmic tRNAs Me t, 3 mitochondria-specific tRNAs Met; 2 can be formylated by the mitochondrial or the E. coli transformylase; all 3 are charged by mitochondrial, chloroplastic or E, coli Met-tRNA synthetase; none is charged by the cytoplasmic enzyme. Chloroplasts contain, in addition to the 2 cytoplasmic tRNAs M e t, 3 chloroplast-specific tRNAs Me t, different from the mitochondrial tRNAs Me t; one is formylatable by the chloroplastic or the E. coli transformylase; all 3 are charged by chloroplastic, mitochondrial or E. coli Met-tRNA synthetase; only one is charged by the cytoplasmic enzyme. Of the 3 E. coli tRNAs Me t, only the formylatable species can be charged by bean cytoplasmic, chloroplastic or mitochondrial Met-tRNA synthetase.

Introduction Preliminary studies from this laboratory had shown the existence of fMett R N A in the chloroplasts [1], etioplasts and mitochondria [2] of Phaseolus vulgaris. Further studies devoted to mitochondria-specific and chloroplast-

241 specific tRNAs M et demonstrated that the chloroplastic tRNAf M et was different from the mitochondrial tRNAf Met [3]. More recently, we have been able to separate the cytoplasmic Met-tRNA synthetase from its chloroplastic counterpart, and we are reporting here the results of experiments where we have studied the aminoacylation of cytoplasmic, chloroplastic and mitochondrial tRNAs Met by homologous and heterologous enzymes. We have also included Escherichia coli tRNAs Met and Met-tRNA synthetase in these comparative studies, in order to check whether tRNAs and enzymes from plant organelles resemble those from bacteria. Material and Methods Chloroplasts were obtained from lyophilized bean leaves by centrifugation in a non-aqueous gradient [4]. Mitochondria were prepared from dark-grown hypocotyls as previously described [2]. Aminoacyl-tRNA synthetases and tRNAs were obtained from chloroplasts, mitochondria or h y p o c o t y l cytoplasm respectively (4). E. coli Met-tRNA synthetase and transformylase were prepared according to Weil [5]. Aminoacylation of tRNAs was performed as previously described [4]. 14C- and 3 H-labelled Met-tRNAs were fractionated by reverse-phase co-chromatography using either the RPC-5 or the RPC-6 system [6,7]. In some cases, RPC-5 chromatography was performed in the presence of 6 M urea pH 3 to achieve a better resolution [3].

z Ix

~o 0 x

A

>' 10

>s _u

W 0

8'0

1 0'0

1 2'0

1; 0

1; 0

1S0

200

Fractions Fig. 1. Fractionation of cytoplasmic and chloroplastic M e t - t R N A synthetases on DEAE-cellulose. A b o u t 10 m g total protein (crude enzymatic preparation) were put on a c o l u m n (10 X 1 c m ) of DEAE-cellulose. Elution was performed as described by Reger [8] using a total v o l u m e of 2 X 2 0 0 ml K C I gradient (0.001--0.4 M). F l o w rate = 20 ml/h. 2-ml fractions were collected and assayed for enzymatic activity. A A c h l o r o p l a s t M e t - t R N A s y n t h e t a s e (as revealed by testing the e n z y m a t i c activity o f the f r a c t i o n s using total chloroplast t R N A ) ; ~ ~, c y t o p l a s m i c M e t - t R N A s y n t h e t a s e (using c y t o p l a s m i c t R N A in the assays).

242 TABLE I A M I N O A C Y L A T I O N O F t R N A Met S P E C I E S F R O M V A R I O U S P L A N T C E L L C O M P A R T M E N T S A N D F R O M E . C O L I BY H O M O L O G O U S A N D H E T E R O L O G O U S E N Z Y M E S T h e results of t h e h e t e r o l o g o u s a m i n o a e y l a t i o n e x p e r i m e n t s are e x p r e s s e d as a p e r c e n t a g e o f t h e a m i n o a c y l a t i o n o b t a i n e d with the s a m e t R N A in t h e c o r r e s p o n d i n g h o m o l o g o u s r e a c t i o n . tRNA

Enzyme

Cytoplasmic Mitochondrial Chloroplastic E. c o l i

Cytoplasmic

Mitochondrial

Chloroplastie

E. coli

100 (1.58)* 30 75 65

58 100 (1.18)* 92 65

62 95 100 (1.12)* 66

40 97 93 100 (2.22)*

* n m o l o f m e t h i o n i n e c h a r g e d p e r m g of t R N A .

Results

(1) Fractionation of the Met-tRNA synthetase from the various cell compartments (a) Fractionation of cytoplasmic and ehloroplastic Met-tRNA synthetase. The two Met-tRNA synthetases were fractionated by DEAE-cellulose chromatography according to Reger et al. [8]. An aliquot of each fraction was used to determine the enzymatic activity. The fractions corresponding to a peak of

2C

b u

1C

~o

~o

~o

~o

~o

~o

~o

~o

Fractions

Fig. 2. R P C - 5 co-chromatography of cytoplasmic t R N A s M e t after aminoacylation with [ 14C] methionine using cytoplasmic M e t - t R N A synthetase (4 4) and of cytoplasmic t R N A s M e t after aminoacylation with [3H] methionine using chloroplastic M e t - t R N A synthetase or mitochondrial M e t - t R N A synthetase or E. coli M e t - t R N A synthetase (~ A). NaCI gradient from 0.25 to 0.32 M (2 × 80 ml) in 6 M urea p H 3. C o l u m n size: 80 × 0.45 cm. Fractions of 2 m l were collected.

243

2C 3 •

1

4 zl

"~ lC

i12

1'0

2o

~o

4'o

5'0

6' 0

' 70

80

Fractions

Fig. 3. R P C - 5 c o - c h r o m a t o g r a p h y o f m i t o e h o n d r i a l t R N A s M e t a f t e r a m i n o a e y l a t i o n w i t h [ 1 4 C ] m e t h i o n i n e u s i n g a m i x t u r e o f c y t o p l a s m i c a n d m i t o c h o n d r i a l M e t - t R N A s y n t h e t a s e (A A), a n d of c y t o p l a s mic tRNAs Met after aminoacylation with [3H]methionine using cytoplasmic Met-tRNA synthetase (~ . . . . . . A). E x p e r i m e n t a l c o n d i t i o n s as in Fig. 2.

2

2C

~z lc

z

~,~

mt L ~ u ~

7 a

4'0 Fractions

Fig. 4. R P C - 5 c o - c h r o m a t o g r a p h y o f m i t o c h o n d r i a l [ 1 4 C ] M e t - t R N A s (A 4) a n d o f m i t o e h o n d r i a l [12 C] f-[ 3 H ] M e t - t R N A s (A . . . . . -~). NaCI g r a d i e n t f r o m 0 . 4 5 t o 0 . 5 0 (2 X 8 0 m l ) in s o d i u m a c e t a t e b u f f e r 0 . 0 1 M p H 4 . 7 , MgC12 0 . 0 1 M. C o l u m n size: 8 0 X 0 . 4 5 era. F r a c t i o n s o f 2 m l w e r e c o l l e c t e d .

244 activity were then pooled, dialyzed against 2 × 2 1 of enzyme buffer [4], concentrated by dialysis against the same buffer containing 50% glycerol and kept at --70 ° C. As shown on Fig. 1, a cytoplasmic preparation of Met-tRNA synthetase yields two peaks of activity (using cytoplasmic tRNA as a substrate). A similar situation exists in the case of bean cytoplasmic Leu-tRNA synthetase which, upon hydroxyapatite chromatography, also gives two peaks which have identical aminoacylation properties [9]. Cytoplasmic Met-tRNA synthetase appears to be very unstable, and in order to obtain the profile shown on Fig. 1, it is necessary to add 10 -s M methionine in all buffers used to prepare and fractionate the enzyme; if kept at --20°C for two weeks the enzymatic activity is lost. This property was used to prepare a chloroplastic Met-tRNA synthetase, free of cytoplasmic Met-tRNA synthetase; such a preparation does indeed show only one peak of activity (using chloroplast t R N A as substrate), which elutes earlier than the two peaks of cytoplasmic Met-tRNA synthetase upon DEAE-cellulose chromatography (Fig. 1) and represents the chloroplast-specific Met-tRNA synthetase (see Table I and Figs 2--5). (b) Attempted fractionation of chloroplastic and mitochondrial MettRNA synthetase. Mitochondrial Met-tRNA synthetase is eluted exactly as its chloroplastic counterpart upon DEAE-cellulose chromatography. The Mett R N A synthetase from the two organelles could not be separated either by chromatography on hydroxyapatite [10] or CM-Sephadex [11]. (2) Aminoacylation of the tRNAs M c t from the various plant cell compartments and from E. coli by homologous and heterologous enzymes The aminoacylation reactions were performed as previously described [~l], at pH 8.2 (which is an optimal pH for plant cytoplasmic, chloroplastic and mitochondrial Met-tRNA synthetase and also for E. coli Met-tRNA synthetase) and using a Mg2+/ATP ratio of 1.0 for cytoplasmic Met-tRNA synthetase, 1.1 for chloroplastic and mitochondrial Met-tRNA synthetase and 3.0 for E. coli Met-tRNA synthetase. The results of these reactions are summarized in Table I. The results of the heterologous aminoacylation experiments are expressed as a percentage of the aminoacylation obtained with the same t R N A in the corresponding homologous reaction. Cytoplasmic tRNAs Me t are only partially aminoacylated by heterologous enzymes. Mitochondrial and chloroplastic tRNAs Met are fully charged by the enzymes of the t w o organelles and of E. coli, whereas E. coli tRNAs Met are only partially aminoacylated by the enzymes from the three plant cell compartments. (a) Aminoacylation o f bean cytoplasmic tRNAs Met. As shown in Fig. 2, when charged by cytoplasmic Met-tRNA synthetase, cytoplasmic tRNAs Met yield two peaks upon RPC-5 chromatography in the presence of 6 M urea at pH 3. But only the first peak can be charged by chloroplastic, mitochondrial or E. coli Met-tRNA synthetase. (b) Aminoacylation o f bean mitochondrial tRNAs Met. Mitochondrial tRNAs Met contain, in addition to the two cytoplasmic peaks (peaks 1 and 4), two mitochondria-specific peaks (peaks 2 and 3) when chromatographed on an

245

RPC-5 column in the presence of 6 M urea at pH 3, after aminoacylation by a mixture of cytoplasmic and mitochondrial Met-tRNA synthetase (Fig. 3). Aminoacylation of peaks 1 and 4 has already been discussed. Mitochondria-specific peaks 2 and 3 can be charged by mitochondrial, chloroplastic or E. coli enzymes, but not by cytoplasmic Met-tRNA synthetase. As shown in our previous studies, the second mitochondria-specific peak (peak 3) can be formylated, using either the mitochondrial or the bacterial transformylase and formyltetrahydrofolate. This peak can be further resolved into two peaks, as shown on Fig. 4, when a shallower gradient is being used (in the absence of urea); both peaks (3a and 3b} can be formylated and then eluted somewhat later, a rather common phenomenon, already discussed by other authors [12] and also observed in our previous studies [3]. Mitochondria thus contain three specific tRNAs Met, namely peaks 2, 3a and 3b. On Fig. 4, cytoplasmic peak 1 is not separated from mitochondrial peak 2 (seen on Fig. 3) and cytoplasmic peak 4 (seen on Fig. 3) is absent because it is not charged by the mitochondrial Met-tRNA synthetase used in these experiments. (c) Aminoacylation of bean chloroplast tRNAs Me t. AS shown on Fig. 5, chloroplastic tRNAs Met contain, in addition to the two cytoplasmic peaks (peaks 1 and 5}, three chloroplast-specific peaks (peaks 2, 3 and 4), when chromatographed on a RPC-5 column in the presence of 6 M urea at pH 3, after aminoacylation by a mixture of cytoplasmic and chloroplastic enzymes and formylation in the presence of formyltetrahydrofolate and transformylase. Peak 2 is the formylatable isoacceptor species; when it is not formylated it elutes together with cytoplasmic peak 1, but elutes later when formylated.

2

t

<-> e '' ' -

o

70

5

2'o

"" . . . . . . . . ~ 'go .4'o

~ do

do

........ - , 70 ao

Fractions

Fig. 5. RPC-5 c o - c h r o m a t o g r a p h y o f c h l o r o p l a s t i c t R N A s Met after a m i n o a c y l a t i o n w i t h [ 14 C] m e t h i o n i n e using a m i x t u z e o f c y t o p l a s m i c and chloroplastic M e t - t R N A s y n t h e t a s e and after f o r m y l a t i o n in the pzesence o f [ 12 C] f o r m y l t e t r a h y d r o f o l a t e (4 A) a n d o f c y t o p l a s m i c t R N A s M e t after a m i n o a c y l a t i o n with [ 3 H] m e t h i o n i n e using c y t o p l a s m i c M e t - t R N A s y n t h e t a s e (z~. . . . . . z0. E x p e r i m e n t a l c o n d i t i o n s as in Fig. 2.

246

4C

1

o w

z~ 20 a,

2

3

• . . . . . . . . ~.__ _~.__.~..__~_...~..__~- . . . . :_.~$ 10

20

30

40

50

60

70

80

F'ractions

F i g . 6. R P C - 6 c o - c h r o m a t o g r a p h y o f E. coli t R N A s M e t a f t e r a m i n o a c y l a t i o n w i t h [ 1 4 C ] m e t h i o n i n e u s i n g E. coli M e t - t R N A s y n t h e t a s e ( a --A) and after aminoacylation with [3H]methionine using cytoplasmic or chloroplastic or mitochondrial M e t - t R N A s y n t h e t a s e (z~. . . . . . z~). N a C l g r a d i e n t f r o m 0 . 3 0 t o 0 . 5 0 M ( 2 X 8 0 m l ) i n s o d i u m a c e t a t e b u f f e r 0 . 0 1 M p H 4 . 7 , M g C l 2 0 . 0 1 M. C o l u m n s i z e : 8 0 X 0 . 4 5 c m . Fractions of 2 ml were collected.

Chloroplast-specific tRNAs Me t 2, 3 and 4 can be aminoacylated by chloroplast, mitochondrial or E. coli enzymes. Only peak 3 can be charged by cytoplasmic Met-tRNA synthetase. (d) Aminoacylation o f E. coli t R N A s Met. As shown on Fig. 6, upon RPC-6 c h r o m a t o g r a p h y , E. coli tRNAs Me t are resolved into 3 peaks, as already r e p o r t e d by Pearson et al. [6]. Only the first one, which is the formylatable initiator species, can be charged by plant cytoplasmic, chloroplastic or mitochondrial enzymes; the initiator tRNAf M e t is know n to represent a b o u t 70% of the E. coli tRNAs Met [ 1 3 , 2 ] , and the fact t h at it is the only species recognized by the three plant enzymes coincides with the results we obtained (65--66%) in our heterologous aminoacylation reactions with E. coli tRNAs M e t (Table I). Discussion Plant tRNAs Me t , have been studied by several research groups and their results are in good agreement with ours. It seems t h a t there are two isoacceptots in plant cytoplasm: one species which incorporates m e t h i o n i n e into the internal position of the p o l y p e p t i d e chains and c a n n o t be aminoacylated by E. coli Mef~tRNA synthetase, and one initiator species which can be aminoacylated by E. coli Met-tRNA synthetase, but c a n n o t be f o r m y l a t e d even when a

247 bacterial transformylase is being used [14--18] in contrast to yeast or animal cytoplasmic initiator t R N A Met, although partial formylation was observed using Anacystis nidulans transformylase [19]. Our studies have shown that this cytoplasmic initiator t R N A Met can be charged not only b y E. coli, b u t also by mitochondrial and chloroplastic Met-tRNA sypthetase. Three tRNAs M ~t have been found in wheat [20,21] and cotton [22--24] chloroplasts, t w o major species and a minor one; the minor one s e e m s t o be a cytoplasmic contaminant; both major species can be charged by chloroplast or E. coli Met~tRNA synthetase, and one of them can be formylated by chloroplast or E. coli transformylase. Our studies have shown the existence of three chloroplast-specific tRNAs Met in bean chloroplasts; they can be charged by chloroplast, mitochondrial or E. coli Met-tRNA synthetase. We have characterized three mitochondria-specific tRNAsMet; two of them are formylatable; all three can be aminoacylated by mitochondrial, chloroplastic or E. coli Met-tRNA synthetase b u t not by cytoplasmic Met-tRNA synthetase. Our studies have shown that out of the three E. coli tRNAs M e t, only the initiator c a n b e aminoacylated by plant cytoplasmic, chloroplastic or mitochondrial Met-tRNA synthetase. Merrick and Dure [24] also observed that E. coli tRNA~ M et was the only species charged by an enzyme extract from c o t t o n cotyledons. Bean cytoplasmic and chloroplastic Met-tRNA synthetases could be separated upon DEAE-cellulose chromatography. In the case of Euglena gracilis, Krauspe and Parthier were able to separate the t w o enzymes b y chromatography on hydroxyapatite [25]. The fact that a given cytoplasmic aminoacylt R N A synthetase differs from its chloroplastic counterpart in its intracellular localization, chromatographic behaviour and t R N A specificity raises the question of the origin of the chloroplastic enzyme; it is n o t known whether it is coded by the chloroplast DNA and synthesized in the organelle, or if it is coded by a nuclear gene, made on cytoplasmic ribosomes and imported into the chloroplasts [26,27]; there is even a possibility that the two enzymes are products of the same gene and that the chloroplastic enzyme is modified (for instance by limited proteolysis) upon entering the organelle. Further studies on cytoplasmic and chloroplastic aminoacyl-tRNA synthetases are necessary to elucidate these problems. Our comparative studies on the tRNAs Leu and Leu-tRNA synthetases in the three cell-compartments of P. vulgaris had suggested the existence of two groups: one consisting of cytoplasmic and mitochondrial tRNAs and enzymes, the other of chloroplastic and E. coli tRNAs and enzymes; cross-aminoacylation reactions were possible within the group b u t n o t between the t w o groups [9]. Our studies on the tRNAs M ~t and the Met-tRNA synthetase in the three compartments show one group consisting of chloroplastic, mitochondrial and E. coli tRNAs and enzymes; cross aminoacylation reactions are possible within this group, whose components clearly differ from cytoplasmic tRNAs Met and Met-tRNA synthetase. These results suggest a certain degree of similarity between bacterial and organellar tRNAs and aminoacyl-tRNA synthetases, and show that tRNAs and enzymes in the organelles often differ from their cytoplasmic counterparts.

248

Acknowledgements We thank Mrs A. Klein for her excellent technical assistance, Mr J. Ramia sa who participated in the last steps of this work, Miss G. Jeannin and Mr A Steinmetz for providing us with purified chloroplast preparations. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

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