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Characterization of Phaseolus vulgaris cytoplasmic, chloroplastic and mitochondrial tRNAsPhe; Aminoacylation by homologous and heterologous enzymes
Plant Science Letters, 13 (1978) 75--81
75
© Elsevier/Nortli-Holland Scientific Publishers Ltd.
CHARACTERIZATION OF P H A S E O L U S V U L G A R I S CYTOPLASMIC, CHLOROPLASTIC AND MITOCHONDRIAL tRNAs Phe; AMINOACYLATION BY HOMOLOGOUS AND HETEROLOGOUS ENZYMES. G. JEANNIN, G. BURKARD and J.H. WEIL Institut de Biologie Mol~culaire et CeUulaire, Universit~ Louis Pasteur, 15 rue Descartes, 6 7084 Strasbourg, (France)
(Received January 6th, 1978) (Accepted February 25th, 1978)
SUMMARY Phaseolus vulgaris tRNAs Phe can be fractionated by reverse phase chromatography into four species: • One cytoplasmic t R N A Phe, which can be aminoacylated by the cytoplasmic phenylalanyl-tRNA synthetase, but not by the chloroplastic, mitochondrial or E. coli enzyme • Two chloroplastic tRNAs Phe, which can be aminoacylated by the chloroplastic, mitochondrial or bacterial enzyme, but not by the cytoplasmic enzyme • One mitochondrial tRNA Phe, which can be charged by the mitochondrial, chloroplastic or bacterial enzyme, but not by the cytoplasmic enzyme. The similarity between organellar and prokaryotic tRNAs Phe suggested by these cross-aminoacylation reactions has recently been confirmed by structure comparisons.
INTRODUCTION The existence of organelle-specific tRNAs and aminoacyl-tRNA synthetases has been well d o c u m e n t e d in the case of plant chloroplasts and mitochondria (for reviews, see refs. 1, 2). Comparative studies on the tRNAs and aminoacyltRNA synthetases present in the 3 cell compartments of Phaseolus vulgaris have led to different conclusions depending on the amino acid tested: In the case of leucine [3], the cytoplasmic and mitochondrial tRNAs can be charged by the cytoplasmic or mitochondrial leucyl-tRNA synthetase, while cross-aminoacylation reactions are possible between chloroplastic and E.coli tRNAs and enzymes. In the case of methionine [4], lysine and proline [5] however, chloroplastic and mitochondrial tRNAs can be aminoacylated by the organellar
76 or bacterial enzyme, but not by the cytoplasmic enzyme, whereas the cytoplasmic tRNAs can be charged by the cytoplasmic enzyme. Because of these conflicting pictures, we decided to investigate the situation in one more case, that of phenylalanine, and we are reporting here the results obtained with bean cytoplasmic, chloroplastic and mitochondrial tRNAs Phe and phenylalanyl-tRNA synthetases. MATERIALS
AND METHODS
Mitochondria were prepared from dark-grown bean hypocotyls as previously described [6]. Chloroplasts were obtained from fresh bean primary leaves essentially as described by Herrmann et al. [7]. Aminoacyl-tRNA synthetases and tRNAs were prepared from mitochondria, chloroplasts or hypocotyl cytoplasm as previously described [8]. E. coli aminoacyl-tRNA synthetases were prepared according to Weft [9]. Aminoacylation of tRNAs was performed as previously described [8], except that HEPES buffer 0.05 M pH 7.5 was used instead of sodium cacodylate. [ 14C] and [ all] phenylalanyl-tRNAs were prepared using phenylalanine with a high specific radioactivity, in the presence of the 19 other unlabeled amino acids, and recovered from the reaction mixture by DEAE-ceUulose chromatography, prior to fractionation by reverse-phase chromatography using the RPC-5 system [10]. The phenylalanyl-tRNA synthetases from the 3 cell compartments were fractionated by chromatography on hydroxyapatite [11]. An aliquot of each fraction was used to determine the enzymatic activity in the presence of cytoplasmic tRNA, of total bean leaf t R N A deprived of cytoplasmic t R N A l'heby Sepharose chromatography [12], or of E. coli tRNA, to reveal the cytoplasmic, chloroplastic, or mitochondrial phenylalanyl-tRNA synthetase respectively. The organellarphenylalanyl-tRNA synthetases are very labileand are rapidly inactivated. RESULTS
Fractionation o f the phenylalanyl-tRNA synthetases from the three cell compartme n ts When a cytoplasmic preparation of phenylalanyl-tRNA synthetase is chromatographed on hydroxyapatite, and when cytoplasmic tRNA is used in the aminoacylation assay to test the enzymatic activity of the fractions, only one peak of activity is observed which is eluted at a concentration of phosphate of about 0.2 M (peak II, Fig. la). When E. coli tRNA is used as a substrate to test the enzymatic activity of the fractions, peak II is not revealed (in other words cytoplasmic phenylalanyl-tRNA synthetase does not charge E. coli tRNAPhe); there is a very low enzymatic activity at the position where peak I (see below) is eluted, suggesting a very low level of contamination of the cytoplasmic preparation by organellar enzyme (it is difficult to avoid mitochondrial contamination when preparing hypocotyl cytoplasm).
77
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t< >N Z 10
20
30
100 fractions
Fig. 1. Fractionation of cytoplasmic, chloroplastic and mitochondrial phenylalanyl-tRNA synthetases. About 40 mg total protein (crude enzymatic preparation) were put on a column (8 × 0.8 cm) of hydroxyapatite. Elution was performed using a total vol of 2 × 30 ml phosphate gradient (0.005--0. 3 M) pH 7.5, MgC12 0.001 M, mercaptoethanol 0.005 M, phenylalanine 0.01 M, glycerol 50%; 0.5 ml fractions were collected and assayed for enzymatic activity. (a) Fractionation of a cytoplasmic enzymatic preparation; (b) Fractionation of a chloroplastic or a mitochondrial enzymatic preparation; Enzyme activity revealed by using total cytoplasmic tRNA • •, E. c o l i tRNA o-- -- -- ---~, chloroplastic tRNA (total bean leaf tRNA minus cytoplasmic tRNA Phe) • A.
When a chloroplastic preparation of phenylalanyl-tRNA synthetase is chromatographed on hydroxyapatite and when chloroplast t R N A Phe is used as a substrate to test the fractions (in fact total bean leaf t R N A deprived of cytoplasmic t R N A Phe was used), one peak of activity is observed (peak I, Fig. l b ) which is eluted at a phosphate concentration of about 0.08 M; but when cytoplasmic t R N A is used as a substrate, another peak of, activity is observed (peak II), which is eluted at a phosphate concentration of about 0.2 M and represents the cytoplasmic phenylalanyl-tRNA synthetase. With a mitochondrial preparation of phenylalanyl-tRNA synthetase, similar
78
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Fig. 2. RPC-5 chromatography of cytoplasmic (a), chloroplastic (b) and mitochondrial (c) phenylalanyl-tRNAs after aminoacylation with homologous or heterologous enzymes. NaC1 gradient from 0.55--0.75 M (2 × 60 ml) pH 4.7; column size 65 × 0.6 cm; fractions of 0.8 ml were collected. Aminoacylation performed with cytoplasmic enzyme preparation or fractionated peak II (see Fig. 1) • , ; aminoacylation performed with organeUar peak I (see Fig. 1) or E. coli phenylalanyl-tRNA synthetase • . . . . • ; aminoacylation performed with total chloroplastic or mitochondrial enzyme (peak I + peak II) o o.
79 results are obtained: when E. coli tRNA is used as a substrate instead of organeUar tRNA (as cross-aminoacylation takes place between E. coli and organellar tRNAs Phe and phenylalanyl-tRNA synthetases), one peak of activity is observed, which is superimposable with peak I obtained for the chloroplastic enzyme; when cytoplasmic tRNA is used as a substrate, the cytoplasmic phenylalanyl-tRNA synthetase (peak II) is also observed (Fig. lb). Characterization and aminoacylation properties of the tRNAs Phe from the three compartments When cytoplasmic tRNA is charged with phenylalanine using either a cytoplasmic enzyme preparation or fractionated peak II (see Fig. la), and submitted to RPC-5 chromatography, one peak is observed (Fig. 2a). This cytoplasmic tRNA Phe is not charged by the chloroplastic, the mitochondrial or the E. coli enzyme. When chloroplast tRNA is charged using either chloroplastic enzyme peak I (Fig. lb) or E. coli enzyme, and submitted to RPC-5 chromatography, 2 peaks of chloroplastic tRNA Phe are observed (Fig. 2b). These 2 peaks are clearly different from the cytoplasmic tRNA Phe also present in this tRNA preparation, but which is only charged when cytoplasmic enzyme is used. That the mitochondrial phenylalanyl-tRNA synthetase is also able to specifically charge the two chloroplastic tRNAs Phe is shown by: (i) the fact that a total mitochondrial enzyme preparation (containing peak I and peak II enzymes) charges the two chloroplastic tRNAs Phe and the cytoplasmic tRNA Phe yielding upon RPC-5 a broad peak which contains all three tRNAs Phe (not shown); (ii) the fact that the mitochondrial enzyme charges a bean leaf tRNA which has been deprived (by Sepharose chromatography) of cytoplasmic tRNA Phe and which therefore contains only the two chloroplastic tRNAs Phe (this aminoacylation is due to mitochondrial peak I enzyme, because peak II does not charge the two chloroplastic tRNAsPhe). When mitochondrial tRNA is charged using E. coli enzyme and submitted to RPC-5 chromatography, one peak of mitochondrial tRNA Phe is observed, which is eluted before the two chloroplastic and the cytoplasmic tRNAs Phe (Fig. 2c). The cytoplasmic enzyme (peak II) does not charge this mitochondrial tRNA Phe . When total chloroplastic or mitochondrial enzymes are used, which contain in fact the organellar (peak I) and the cytoplasmic (peak II) enzymes, two peaks are observed upon RPCo5 chromatography because both the mitochondria-specific and the cytoplasmic tRNAs Phe have been aminoacylated. DISCUSSION Phaseolus vulgaris phenylalanyl-tRNA synthetases can be separated by hydroxyapatite chromatography [11, 13] into two species : a cytoplasmic enzyme which is eluted at a phosphate concentration of about 0.2 M (peak II, Fig. 1), and an organellar enzyme which is eluted at a phosphate concentration of about 0.08 M (peak I). Peak I enzyme is present in both chloroplasts and mitochondria,
80 but it is not known whether the same enzyme is present in the two organelles or if there are two different enzymes. Upon RPC-5 chromatography, Phaseolus vulgaris tRNAs Phe can be separated into 4 species: • One cytoplasmic tRNA Phe, which can be aminoacylated by the cytoplasmic phenylalanyl-tRNA synthetase, but not by the chloroplastic, mitochondrial or E. coli enzyme. This species is eluted early upon Sepharose chromatography using a reverse salt gradient [ 12 ], a property we have used in this study to deprive bean leaf total tRNA of cytoplasmic tRNA Phe, but is strongly retained on BD-cellulose and is eluted only by addition of ethanol [14] because of the Y base present in this t R N A Phe. • Two chloroplastic tRNAs Phe, which can be aminoacylated by the chloroplastic, mitochondrial or bacterial enzyme, but not by the cytoplasmic enzyme. Upon BD-cellulose chromatography, two chloroplastic tRNAs Phe are also obtained [ 14], and the nucleotide sequence of the major chloroplastic species has recently been determined [15]. These two chloroplastspecific tRNAs Phe hybridize to chloroplast DNA and are therefore coded by the chloroplast genome [16]. • One mitochondrial tRNA Phe, which can be aminoacylated by the mitochondrial, chloroplastic or bacterial enzyme, but not by the cytoplasmic enzyme. The results of our heterologous aminoacylation experiments show that bean chloroplastic and mitochondrial tRNAs Phe can be charged by the chloroplastic, mitochondrial or bacterial phenyialanyl-tRNA synthetase, but not by the cytoplasmic enzyme, whereas the cytoplasmic t R N A Phe can only be aminoacylated by the cytoplasmic enzyme and not by the organeUar or bacterial enzyme. A similar situation has been observed in the case of bean tRNAs Met [4], tRNAs Lys and tRNAs Pr° [5]. The similarity between organellar and prokaryotic tRNAs Phe suggested by these cross-aminoacylation reactions (they are recognized by the same enzymes) is confirmed by structure comparisons: if one considers the 48 nucleotides c o m m o n to all prokaryotic tRNAs Phe which have been sequenced, only 3 are different in bean chloroplast tRNA Phe [15]. A similarity between chloroplastic and prokaryotic tRNAs and aminoacylt R N A synthetases is also suggested by cross-aminoacylation reactions performed between cotton chloroplasts and E. coli [17] and between the blue alga Anacystis nidulans, E. coli and Euglena chloroplasts [18, 19], but mitochondrial tRNAs and enzymes were not included in these studies. In contrast to the situation observed in the case of methionine, lysine, proline and phenylalanine, there is for leucine a similarity between bean cytoplasmic and mitochondrial tRNAs and enzymes on one hand, and between chloroplastic and E. coil tRNAs and enzymes on the other hand [3]. Such a resemblance between cytoplasmic and mitochondrial aminoacyl-tRNA synthetases has also been reported for leucine in tobacco [20] and for isoleucine in Euglena [21]. Also in Euglena, chloroplast phenylalanyl-tRNA synthetase (enzyme I activity
81
eluting from hydroxyapatite column at 0.05 M phosphate) was found to charge both prokaryotic and cytoplasmic tRNAs [22]. This relative unspecificity in aminoacylation appeared to be due to the ambiguous charging ability of the chloroplast enzyme, rather than to the existence of 2 enzymes with the same chromatographic properties, although the presence in fraction I of another (mitochondrial) enzyme activity could not be excluded. In any case, Euglena and Phaseolus chloroplast phenylalanyl-tRNA synthetases appear to differ in their aminoacylation specificity, while the two chloroplast tRNAs Phe are very similar [15]. ACKNOWLEDGEMENTS
This work was supported by a grant from the Commissariat ~ rEnergie Atomique. REFERENCES 1 J.H. Weil, G. Burkard, P. Guillemaut, G. Jeannin, R. Martin and A. Steinmetz in L. Bogorad and J.H. Well (Eds) Nucleic acids and protein synthesis in plants, Plenum Press (New York) 1977, pp. 97--120. 2 W.E. Barnett, S.D. Schwartzbach and L.I. Hecker, Prog. Nucl. Acid Res. Mol. Biol., 21 (1978) in press. 3 P. Guillemaut, P. Steinmetz, G. Burkard and J.H. Weil, Biochim. Biophys. Acta, 378 (1975) 64. 4 P. Guillemaut and J.H. Weil, Biochim. Biophys. Acta, 407 (1975) 240 5 G. Jeannin, G. Burkard and J.H. Weil, Biochim. Biophys. Acta, 442~1976) 24. 6 P. Guillemaut, G. Burkard and J.H. Weil, Phytochemistry, 11 (1972) 2217. 7 R.G. Herrmann, H.J. Bohnert, K.V. Kowallik and J.M. Schmitt, Biochim. Biophys. Acta, 378 (1975) 305. 8 G. Burkard, P. Guillemaut and J.H. Weil, Biochim. Biophys. Acta, 224 (1970) 184. 9 J.H. Weil, Bull. Soc. Chim. Biol., 51 (1969) 1479. 10 R.L. Pearson, J.F. Weiss and A.D. Kelmers, Biochim. Biophys. Acta, 228 (1971) 770. 11 B.J. Reger, S.A. Fairfield, J.L. Epler and W.E. Barnett, Proc. Natl. Acad. Sci., US 67 (1970) 1207. 12 W.M. Holmes, R.E. Hurd, B.R. Reid, R.A. Rimerman and G.W. Hatfield, Proc. Natl. Acad. Sci., U.S.A. 72 (1975) 1068. 13 R. Krauspe and B. Parthier, Biochem. Physiol. Pflanz, 165 (1974) 18. 14 P. Guillemaut, R. Martin and J.H. Weil, FEBS Lett., 63 (1976) 273. 15 P. Guillemaut and G. Keith, FEBS Lett., 84 (1977) 351. 16 A. Steinmetz and J.H. Weil, Biochim. Biophys. Acta, 454 (1976) 429. 17 W.C. Merrick and L.S. Dure, Biochemistry, 12 (1973) 629. 18 N. Beauchemin, B. Lame and R.J. Cedergren, Arch. Biochem. Biophys., 156 (1973) 17. 19 B. Parthier and R. Krauspe, Biochem. Physiol. Pflanz, 165 (1974) 1. 20 R.H. Guderian, R.L. Pulliam and H.P. Gordan, Biochim. Biophys. Acta, 262 (1972) 50. 21 U. Kislev, M.I. Selsky, C. Norton and J.M. Eisenstadt, Biochim. Biophys. Acta, 287 (1972) 256. 22 B. Parthier and R. Krauspe, Plant Sci. Lett., 1 (1973) 211.
75
© Elsevier/Nortli-Holland Scientific Publishers Ltd.
CHARACTERIZATION OF P H A S E O L U S V U L G A R I S CYTOPLASMIC, CHLOROPLASTIC AND MITOCHONDRIAL tRNAs Phe; AMINOACYLATION BY HOMOLOGOUS AND HETEROLOGOUS ENZYMES. G. JEANNIN, G. BURKARD and J.H. WEIL Institut de Biologie Mol~culaire et CeUulaire, Universit~ Louis Pasteur, 15 rue Descartes, 6 7084 Strasbourg, (France)
(Received January 6th, 1978) (Accepted February 25th, 1978)
SUMMARY Phaseolus vulgaris tRNAs Phe can be fractionated by reverse phase chromatography into four species: • One cytoplasmic t R N A Phe, which can be aminoacylated by the cytoplasmic phenylalanyl-tRNA synthetase, but not by the chloroplastic, mitochondrial or E. coli enzyme • Two chloroplastic tRNAs Phe, which can be aminoacylated by the chloroplastic, mitochondrial or bacterial enzyme, but not by the cytoplasmic enzyme • One mitochondrial tRNA Phe, which can be charged by the mitochondrial, chloroplastic or bacterial enzyme, but not by the cytoplasmic enzyme. The similarity between organellar and prokaryotic tRNAs Phe suggested by these cross-aminoacylation reactions has recently been confirmed by structure comparisons.
INTRODUCTION The existence of organelle-specific tRNAs and aminoacyl-tRNA synthetases has been well d o c u m e n t e d in the case of plant chloroplasts and mitochondria (for reviews, see refs. 1, 2). Comparative studies on the tRNAs and aminoacyltRNA synthetases present in the 3 cell compartments of Phaseolus vulgaris have led to different conclusions depending on the amino acid tested: In the case of leucine [3], the cytoplasmic and mitochondrial tRNAs can be charged by the cytoplasmic or mitochondrial leucyl-tRNA synthetase, while cross-aminoacylation reactions are possible between chloroplastic and E.coli tRNAs and enzymes. In the case of methionine [4], lysine and proline [5] however, chloroplastic and mitochondrial tRNAs can be aminoacylated by the organellar
76 or bacterial enzyme, but not by the cytoplasmic enzyme, whereas the cytoplasmic tRNAs can be charged by the cytoplasmic enzyme. Because of these conflicting pictures, we decided to investigate the situation in one more case, that of phenylalanine, and we are reporting here the results obtained with bean cytoplasmic, chloroplastic and mitochondrial tRNAs Phe and phenylalanyl-tRNA synthetases. MATERIALS
AND METHODS
Mitochondria were prepared from dark-grown bean hypocotyls as previously described [6]. Chloroplasts were obtained from fresh bean primary leaves essentially as described by Herrmann et al. [7]. Aminoacyl-tRNA synthetases and tRNAs were prepared from mitochondria, chloroplasts or hypocotyl cytoplasm as previously described [8]. E. coli aminoacyl-tRNA synthetases were prepared according to Weft [9]. Aminoacylation of tRNAs was performed as previously described [8], except that HEPES buffer 0.05 M pH 7.5 was used instead of sodium cacodylate. [ 14C] and [ all] phenylalanyl-tRNAs were prepared using phenylalanine with a high specific radioactivity, in the presence of the 19 other unlabeled amino acids, and recovered from the reaction mixture by DEAE-ceUulose chromatography, prior to fractionation by reverse-phase chromatography using the RPC-5 system [10]. The phenylalanyl-tRNA synthetases from the 3 cell compartments were fractionated by chromatography on hydroxyapatite [11]. An aliquot of each fraction was used to determine the enzymatic activity in the presence of cytoplasmic tRNA, of total bean leaf t R N A deprived of cytoplasmic t R N A l'heby Sepharose chromatography [12], or of E. coli tRNA, to reveal the cytoplasmic, chloroplastic, or mitochondrial phenylalanyl-tRNA synthetase respectively. The organellarphenylalanyl-tRNA synthetases are very labileand are rapidly inactivated. RESULTS
Fractionation o f the phenylalanyl-tRNA synthetases from the three cell compartme n ts When a cytoplasmic preparation of phenylalanyl-tRNA synthetase is chromatographed on hydroxyapatite, and when cytoplasmic tRNA is used in the aminoacylation assay to test the enzymatic activity of the fractions, only one peak of activity is observed which is eluted at a concentration of phosphate of about 0.2 M (peak II, Fig. la). When E. coli tRNA is used as a substrate to test the enzymatic activity of the fractions, peak II is not revealed (in other words cytoplasmic phenylalanyl-tRNA synthetase does not charge E. coli tRNAPhe); there is a very low enzymatic activity at the position where peak I (see below) is eluted, suggesting a very low level of contamination of the cytoplasmic preparation by organellar enzyme (it is difficult to avoid mitochondrial contamination when preparing hypocotyl cytoplasm).
77
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I
"iT
,..
~.
I~:
0.3
uZI
•
0.2
~
0.1
E
t< >N Z 10
20
30
100 fractions
Fig. 1. Fractionation of cytoplasmic, chloroplastic and mitochondrial phenylalanyl-tRNA synthetases. About 40 mg total protein (crude enzymatic preparation) were put on a column (8 × 0.8 cm) of hydroxyapatite. Elution was performed using a total vol of 2 × 30 ml phosphate gradient (0.005--0. 3 M) pH 7.5, MgC12 0.001 M, mercaptoethanol 0.005 M, phenylalanine 0.01 M, glycerol 50%; 0.5 ml fractions were collected and assayed for enzymatic activity. (a) Fractionation of a cytoplasmic enzymatic preparation; (b) Fractionation of a chloroplastic or a mitochondrial enzymatic preparation; Enzyme activity revealed by using total cytoplasmic tRNA • •, E. c o l i tRNA o-- -- -- ---~, chloroplastic tRNA (total bean leaf tRNA minus cytoplasmic tRNA Phe) • A.
When a chloroplastic preparation of phenylalanyl-tRNA synthetase is chromatographed on hydroxyapatite and when chloroplast t R N A Phe is used as a substrate to test the fractions (in fact total bean leaf t R N A deprived of cytoplasmic t R N A Phe was used), one peak of activity is observed (peak I, Fig. l b ) which is eluted at a phosphate concentration of about 0.08 M; but when cytoplasmic t R N A is used as a substrate, another peak of, activity is observed (peak II), which is eluted at a phosphate concentration of about 0.2 M and represents the cytoplasmic phenylalanyl-tRNA synthetase. With a mitochondrial preparation of phenylalanyl-tRNA synthetase, similar
78
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10
20
30
40
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150
Fig. 2. RPC-5 chromatography of cytoplasmic (a), chloroplastic (b) and mitochondrial (c) phenylalanyl-tRNAs after aminoacylation with homologous or heterologous enzymes. NaC1 gradient from 0.55--0.75 M (2 × 60 ml) pH 4.7; column size 65 × 0.6 cm; fractions of 0.8 ml were collected. Aminoacylation performed with cytoplasmic enzyme preparation or fractionated peak II (see Fig. 1) • , ; aminoacylation performed with organeUar peak I (see Fig. 1) or E. coli phenylalanyl-tRNA synthetase • . . . . • ; aminoacylation performed with total chloroplastic or mitochondrial enzyme (peak I + peak II) o o.
79 results are obtained: when E. coli tRNA is used as a substrate instead of organeUar tRNA (as cross-aminoacylation takes place between E. coli and organellar tRNAs Phe and phenylalanyl-tRNA synthetases), one peak of activity is observed, which is superimposable with peak I obtained for the chloroplastic enzyme; when cytoplasmic tRNA is used as a substrate, the cytoplasmic phenylalanyl-tRNA synthetase (peak II) is also observed (Fig. lb). Characterization and aminoacylation properties of the tRNAs Phe from the three compartments When cytoplasmic tRNA is charged with phenylalanine using either a cytoplasmic enzyme preparation or fractionated peak II (see Fig. la), and submitted to RPC-5 chromatography, one peak is observed (Fig. 2a). This cytoplasmic tRNA Phe is not charged by the chloroplastic, the mitochondrial or the E. coli enzyme. When chloroplast tRNA is charged using either chloroplastic enzyme peak I (Fig. lb) or E. coli enzyme, and submitted to RPC-5 chromatography, 2 peaks of chloroplastic tRNA Phe are observed (Fig. 2b). These 2 peaks are clearly different from the cytoplasmic tRNA Phe also present in this tRNA preparation, but which is only charged when cytoplasmic enzyme is used. That the mitochondrial phenylalanyl-tRNA synthetase is also able to specifically charge the two chloroplastic tRNAs Phe is shown by: (i) the fact that a total mitochondrial enzyme preparation (containing peak I and peak II enzymes) charges the two chloroplastic tRNAs Phe and the cytoplasmic tRNA Phe yielding upon RPC-5 a broad peak which contains all three tRNAs Phe (not shown); (ii) the fact that the mitochondrial enzyme charges a bean leaf tRNA which has been deprived (by Sepharose chromatography) of cytoplasmic tRNA Phe and which therefore contains only the two chloroplastic tRNAs Phe (this aminoacylation is due to mitochondrial peak I enzyme, because peak II does not charge the two chloroplastic tRNAsPhe). When mitochondrial tRNA is charged using E. coli enzyme and submitted to RPC-5 chromatography, one peak of mitochondrial tRNA Phe is observed, which is eluted before the two chloroplastic and the cytoplasmic tRNAs Phe (Fig. 2c). The cytoplasmic enzyme (peak II) does not charge this mitochondrial tRNA Phe . When total chloroplastic or mitochondrial enzymes are used, which contain in fact the organellar (peak I) and the cytoplasmic (peak II) enzymes, two peaks are observed upon RPCo5 chromatography because both the mitochondria-specific and the cytoplasmic tRNAs Phe have been aminoacylated. DISCUSSION Phaseolus vulgaris phenylalanyl-tRNA synthetases can be separated by hydroxyapatite chromatography [11, 13] into two species : a cytoplasmic enzyme which is eluted at a phosphate concentration of about 0.2 M (peak II, Fig. 1), and an organellar enzyme which is eluted at a phosphate concentration of about 0.08 M (peak I). Peak I enzyme is present in both chloroplasts and mitochondria,
80 but it is not known whether the same enzyme is present in the two organelles or if there are two different enzymes. Upon RPC-5 chromatography, Phaseolus vulgaris tRNAs Phe can be separated into 4 species: • One cytoplasmic tRNA Phe, which can be aminoacylated by the cytoplasmic phenylalanyl-tRNA synthetase, but not by the chloroplastic, mitochondrial or E. coli enzyme. This species is eluted early upon Sepharose chromatography using a reverse salt gradient [ 12 ], a property we have used in this study to deprive bean leaf total tRNA of cytoplasmic tRNA Phe, but is strongly retained on BD-cellulose and is eluted only by addition of ethanol [14] because of the Y base present in this t R N A Phe. • Two chloroplastic tRNAs Phe, which can be aminoacylated by the chloroplastic, mitochondrial or bacterial enzyme, but not by the cytoplasmic enzyme. Upon BD-cellulose chromatography, two chloroplastic tRNAs Phe are also obtained [ 14], and the nucleotide sequence of the major chloroplastic species has recently been determined [15]. These two chloroplastspecific tRNAs Phe hybridize to chloroplast DNA and are therefore coded by the chloroplast genome [16]. • One mitochondrial tRNA Phe, which can be aminoacylated by the mitochondrial, chloroplastic or bacterial enzyme, but not by the cytoplasmic enzyme. The results of our heterologous aminoacylation experiments show that bean chloroplastic and mitochondrial tRNAs Phe can be charged by the chloroplastic, mitochondrial or bacterial phenyialanyl-tRNA synthetase, but not by the cytoplasmic enzyme, whereas the cytoplasmic t R N A Phe can only be aminoacylated by the cytoplasmic enzyme and not by the organeUar or bacterial enzyme. A similar situation has been observed in the case of bean tRNAs Met [4], tRNAs Lys and tRNAs Pr° [5]. The similarity between organellar and prokaryotic tRNAs Phe suggested by these cross-aminoacylation reactions (they are recognized by the same enzymes) is confirmed by structure comparisons: if one considers the 48 nucleotides c o m m o n to all prokaryotic tRNAs Phe which have been sequenced, only 3 are different in bean chloroplast tRNA Phe [15]. A similarity between chloroplastic and prokaryotic tRNAs and aminoacylt R N A synthetases is also suggested by cross-aminoacylation reactions performed between cotton chloroplasts and E. coli [17] and between the blue alga Anacystis nidulans, E. coli and Euglena chloroplasts [18, 19], but mitochondrial tRNAs and enzymes were not included in these studies. In contrast to the situation observed in the case of methionine, lysine, proline and phenylalanine, there is for leucine a similarity between bean cytoplasmic and mitochondrial tRNAs and enzymes on one hand, and between chloroplastic and E. coil tRNAs and enzymes on the other hand [3]. Such a resemblance between cytoplasmic and mitochondrial aminoacyl-tRNA synthetases has also been reported for leucine in tobacco [20] and for isoleucine in Euglena [21]. Also in Euglena, chloroplast phenylalanyl-tRNA synthetase (enzyme I activity
81
eluting from hydroxyapatite column at 0.05 M phosphate) was found to charge both prokaryotic and cytoplasmic tRNAs [22]. This relative unspecificity in aminoacylation appeared to be due to the ambiguous charging ability of the chloroplast enzyme, rather than to the existence of 2 enzymes with the same chromatographic properties, although the presence in fraction I of another (mitochondrial) enzyme activity could not be excluded. In any case, Euglena and Phaseolus chloroplast phenylalanyl-tRNA synthetases appear to differ in their aminoacylation specificity, while the two chloroplast tRNAs Phe are very similar [15]. ACKNOWLEDGEMENTS
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