Interactions of aminoacyl-tRNA synthetases in high-molecular-weight multienzyme complexes from rat liver

Biochimica etBiophysica Acta 829 (1985) 319-326 Elsevier

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Interactions of aminoacyl-tRNA synthetases in high-molecular-weight multienzyme complexes from rat liver Chi V. Dang a, Blair Ferguson b,,, Deborah Johnson Burke b,**, Victor Garcia b and David C.H. Yang b.*** a Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, AID 21205 and b Department of Chemistry, Georgetown University, Washington, DC 20057 (U.S.A.) (Received November 12th, 1984)

Key words: Aminoacyl-tRNA synthetase; Multienzyme complex; Enzyme-enzyme interaction; Inactivation kinetics

The functional interaction of Arg-, lie-, Leu-, Lys- and Met-tRNA synthetases occurring within the same rat liver multienzyme complex are investigated by examining the enzymes catalytic activities and inactivation kinetics. The Michaelis constants for amino acids, ATP and tRNAs of the dissociated aminoacyl-tRNA synthetases are not significantly different from those of the high-Mr multienzyme complex, except in a few cases where the K m values of the dissociated enzymes are higher than those of the high-Mr form. The maximal aminoacylation velocities of the individual aminoacyi-tRNA synthetases are not affected by the presence of simultaneous aminoacylation by another synthetase occurring within the same multienzyme complex. Site-specific oxidative modification by ascorbate and nonspecific thermal inactivation of synthetases in the purified rat liver 18 S synthetase complex are examined. Lys- and Arg-tRNA synthetases show remarkably parallel time-courses in both inactivation processes. Leu- and Met-tRNA synthetases also show parallel kinetics in thermal inactivation and possibly oxidative inactivation. Iie-tRNA synthetase shows little inactivation in either process. The oxidative inactivation of Lys- and Arg-tRNA synthetases can be reversed by addition of dithiothreitol. These results suggest that synthetases within the same high-Mr complex catalyze aminoacylation reactions independently; however, the stabilities of some of the synthetases in the multienzyme complex are coupled. In particular, the stability of Arg-tRNA synthetase depends appreciably on its association with fully active Lys-tRNA synthetase.

Introduction

Aminoacyl-tRNA synthetases are a family of enzymes which catalyze the aminoacylation of spceific tRNA during protein biosynthesis [1] and are capable of synthesizing diadenosine-5',5"-

* Present address: Smith, Klein & French Laboratories, Philadelphia, PA 19101, U.S.A. ** Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, U.S.A. *** To whom correspondence should be addressed.

P1,p4-tetraphosphate, a recently recognized intracellular pleiotypic signal dinucleotide [2,3]. Several mammalian aminoacyl-tRNA synthetases occur in high-M r complexes [4,5] in contrast to lower eukaryotic [6] or prokaryotic synthetases [1]. The high-M r synthetases have been highly purified [7-9] and shown to be heterotypic multienzyme complexes [4,10,11]. The polypeptide composition and structural organization of these complexes are now better understood. Methionyl-tRNA synthetase, which occurs in the high-M r complex, was localized intracellularly to the rough endoplasmic reticulum by immunofluorescence microscopy [12].

0167-4838/85/$03.00 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

320 The functional significance of these muhienzyme complexes remains to be explored. Complex formation has been suggested to reduce thermolability of Lys- and Leu-tRNA synthetases [13,14]. Coordinate control of synthetase activities in highM r complexes by phosphorylation has been suggested but not confirmed [15]. Differences in size distribution (allotropism) of high-Mr synthetases have been shown between synthetases from hepatoma and normal liver [16,17]. In this communication, we examine the possibility of protein-protein interactions and altered enzymatic properties in the high-Mr synthetase complexes. High-M r complex formation raises the possibility of interactions between the synthetases and may impart different functional properties to the complexed synthetases as compared to their unassociated counterparts. The ability to purify rat liver Lys-tRNA synthetase dissociated from the high-Mr complex demonstrated that complex formation is not required for the expression of LystRNA synthetase activity [18]. We take advantage of the ability to obtain purified Lys-tRNA synthetase and isolate other unassociated synthetases [18,19] to compare their properties with those found in the high-Mr complexes. To determine whether complex formation of the rat liver aminoacyl-tRNA synthetases may alter the substrate relative affinity toward these enzymes, the Michaelis constants for amino acid, ATP, and tRNA of unassociated and complexed Arg-, Ile-, Leu-, Lys- and Met-tRNA synthetases were compared. Possible catalytic interactions between the synthetases were assessed by comparison of the individual activities in the absence or presence of simultaneous aminoacylation by another synthetase occurring within the same multienzyme complex. The protein-protein interactions among the synthetases in the multienzyme complex may mutually stabilize the component synthetases. Such interactions may be revealed by analyzing the inactivation kinetics of individual synthetase activities. The high-Mr Lys-tRNA synthetase was previously shown to be more thermostable than the dissociated form [13], probably due to the intermolecular interaction among the synthetases. Here, we extend the study to other synthetases in an effort to elucidate the protein-protein interaction among the synthetases in the corn-

plex. Oxidative modification of proteins by ascorbate [20] was also used due to its site specificity. Materials and Methods

Materials. Materials were as described previously [7,18]. Unfractionated tRNA was purified from rat liver as described [19]. Amino acid accepting activities (pmol) in one A260 unit of tRNA were: Arg, 50; Ile, 70; Leu, 150; Lys, 140; Met, 40. Radioactive 14C-labeled amino acids were obtained from New England Nuclear (Boston, MA). Preparation of aminoacyl-tRNA synthetases. The preparation of the 24 S rat liver aminoacyl-tRNA synthetase complex (Fig. 1A) and the dissociation of the 24 S complex by hydrophobic chromatography were as described [18,19]. The unassociated 6 S enzymes were obtained by sucrose gradient centrifugation of the dissociated 24 S complex [18,19]. Fig. 1A shows the isolation of 6 S LystRNA synthetase by sucrose gradient centrifugation. The 18 S rat liver complex was purified as described [7]. The 18 S complex polypeptide composition is shown in Fig. lB. The polypeptide M r ( x 10 -3) was reported previously [7]: band I, 160; band II, 150; band III, 135; band IV, 104; band V, 92; band VI, 69; band VII, 67; and band VIII, 48. The complex contains synthetase activities for Arg, Gln, Ile, Leu, Lys and Met [7]. A nearly identical complex from rabbit liver also contains GIu-tRNA synthetase [8]. The integrity of the 18 S complex was maintained in the presence of substrates as determined by sucrose gradient centrifugation confirming the results of Mirande et al. [21]. The rat liver Lys-tRNA synthetase was purified from dissociated high-Mr complex as described [18], and the polypeptide composition is shown in Fig. lB. Studies in this communication do not include Glu- or GIn-tRNA synthetases, due to their low and unstable activities. Determination of Michaelis constants for tRNA, A TP and amino acids. Michaelis constants were determined for the 24 S complex and partially purified 6 S aminoacyl-tRNA synthetases. Initial velocities were determined by incubation of the standard reaction mixture with saturating amounts of substrates except for the variable substrate (125

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/~1 total volume; 1 m g / m l bovine serum albumin, 100 mM Tris-HC1 (pH 7.5 at 37°C), 25 mM KCI, 3 mM magnesium acetate, 20 mM 2-mercaptoethanol, 2 mM ATP, 0.2 mg unfractionated rat liver tRNA (five .,1260 units), and 0.1 mM 14Clabelled amino acid) in the presence of a limiting amount of enzyme. Aliquots of 25 btl were taken at 0, 2.5, 5.0 and 7.5 min at 37°C and processed as described [19]. All assays were linear with time. All K m values were determined immediately after the preparation of fresh 6 S and 24 S aminoacyl-tRNA synthetases. Influence of non-cognate amino acid on high-Mr synthetase activities. Each synthetase activity in the purified 18 S rat liver complex was determined in the absence and presence of each of the non-cognate amino acid (130/~M pH 7.5) under standard assay conditions (see above). Oxidative modification of aminoacyl-tRNA synthetases. Inactivation by oxidative modification was

carried out according to Levine et al. [20] with modifications. Purified 18 S aminoacyl-tRNA synthetase complex (at 25 units/ml for Lys-tRNA synthetase) was incubated at 37°C with 100 mM Tris-HCl (pH 7.5), 3 mM MgCI 2, 1 m g / m l bovine serum albumin, 10% glycerol and 10 mM ascorbate. The reaction was started by the addition of 0.004 ml of 1 M ascorbate to 0.4 ml of enzyme solution. Ascorbate solution was prepared by neutralizing ascorbic acid (Baker) with 1 M K O H and adding freshly prepared FeCI 3 immediately before use to 2 mM. At time intervals, 0.025 ml samples were taken directly into the standard synthetase assay mix. 1 M dithiothreitol was added to the inactivation mix to 20 mM at 65 min after the addition of ascorbate to reverse the oxidation of sulfhydryl groups. Thermal inactivation of aminoacyl-tRNA synthetases. Inactivation was carried out as previously described [13] with modifications. Purified 18 S

322

synthetase complex (at 35 units/ml for Lys-tRNA synthetase) was incubated with 100 mM Tris-HCl (pH 7.5) and 2 mM dithiothreitol. The inactivation was initiated by transferring the enzyme solution at 0°C to 37°C immediately after dilution. At time intervals, aliquots of 25 ~tl were taken directly into standard synthetase assay mix for 2 min in the presence of 1 m g / m l bovine serum albumin at 37°C. Results

Functional independence of individual synthetases in the synthease complex Unassociated aminoacyl-tRNA synthetases were dissociated from the high-M r complex by hydrophobic chromatography and further purified by sucrose gradient centrifugation [18,19]. Fig. 1A demonstrates the preparative sucrose gradients of 24 S and 6 S rat liver Lys-tRNA synthetase. Yields of unassociated lie- and Arg-tRNA synthetases were low after hydrophobic chromatography [19]; thus, significant contamination by 10-12 S enzymes specific for Ile and Arg cannot be ruled out. The 24 S and 6 S aminoacyl-tRNA synthetases were dialyzed against the same buffer prior to kinetic studies. Michaelis constants were determined for Arg-, lie-, Leu-, Lys- and Met-tRNA synthetases. Michaelis constants for amino acids, ATP and tRNAs were determined for the five synthetase activities and are shown in Table I. The g m values for tRNA are corrected for the specific tRNAs. In most cases, no appreciable differences between the

Michaelis constants of the unassociated 6 S and complexed 24 S enzymes were observed. In a few c a s e s , K m values were significantly different; the dissociated enzymes usually showed higher K m values than the complexed enzymes. Conformational changes during dissociation of these enzymes cannot be ruled out in these cases. Under standard assay conditions, only one amino acid was incubated with the high-M r synthetase complex in the presence of saturating concentrations of unfractionated tRNA and ATP. To test the hypothesis that simultaneous aminoacylation by another synthetase may affect activities of synthetases in the same multienzyme complex, we compared the synthetase activities in the presence and absence of a second amino acid. Changes in Vmax due to simultaneous aminoacylation may be determined. Table II shows no significant (less than 10%) changes in any of the high-M r synthetase activities in the presence of any one of the second amino acids. The results show no significant alteration in Vmax of any synthetase in presence of simultaneous aminoacylation by another synthetase within the same multienzyme complex.

Oxidative inactivation of the high-Mr aminoacyltRNA synthetases The possible protein-protein interaction among the aminoacyl-tRNA synthetases was further investigated by oxidative modification of the purified 18 S rat liver complex. Oxidative inactivation of proteins using ascorbate mimics the action of mixed function oxidation [22]. This system specifically oxidizes an essential histidine residue and

TABLE I M1CHAELIS CONSTANTS FOR 6 S A N D 24 S AMINOACYL-tRNA SYNTHETASES (RS) Experimental conditions are described in detail under Materials and Methods Synthetase

Arg-RS Lys-RS " Ile-RS Leu-RS Met-RS

tRNA K m (/.tM)

Amino acid K m (/.tM)

ATP K m (mM)

6S

24S

6S

24S

6S

24S

0.29 0.57 0.47 1.1 2.5

0.31 0.46 0.16 1.3 1.5

1.8 2.2 2.2 9.1 5.0

1,4 1.1 1.7 8.5 2.4

0.15 0,03 0,07 1.1 0.25

0.15 0.02 0.05 0.34 0.25

a Assay conditions and kinds of enzyme preparations are different from those in Ref. 18.

323 T A B L E II E F F E C T OF A D D I T I O N O F N O N - C O G N A T E A M I N O A C I D ON SYNTHETASE ACTIVITIES A m i n o acids (130/~M)

Aminoacyl-tRNA synthetase (RS) activity (%) Lys-RS

Arg-RS

Leu-RS

Ile-RS

Let-RS

None Lysine Arginine Leucine lsoleucine Methionine

100 104 103 97 104

100 112 110 101 107

100 95 96 108 96

100 101 102 99 100

100 96 100 101 95 -

some as yet unidentified residues of Escherichia coli. glutamine synthetase, rendering it more susceptible to proteolysis [23].

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Fig. 2. (a) Oxidative inactivation of purified 18 S rat liver high-Mr aminoacyl-tRNA synthetases by ascorbate. D T T represents the addition of dithiothreitol (20 m M final concentrations). The abscissa represents the time of incubation of the synthetase complex with ascorbate and Fe 3+. Synthetase activities: Arg, O ©; lie, • O; Leu, [] Q; Lys, I I ; and Met, zx zx. (For experimental details, see Materials and Methods). (b) Thermal inactivation of purified rat liver 18 S aminoacyl-tRNA synthetases. Synthetase activities are for Arg ( O ©), lie (O O), Leu (m D), Lys ( I I ) , and Met (zx zx). The abscissa represents the incubation time at 37°C, and the ordinate represents the percentage of initial activity. The inset shows the comparison of rat liver 24 S and 6 S Lys-tRNA synthetase inactivation kinetics at 37°C; the abscissa represents the incubation time in minutes, and the ordinate represents the percent of initial activity. (For experimental details, see Materials and Methods.)

324

the presence of bovine serum albumin and glycerol to prevent thermal inactivation which was less than 5% for all five activities (data not shown). Lys- and Arg-tRNA synthetases were inactivated by ascorbate in parallel time-courses, and both inactivated enzymes can be reactivated by addition of dithiothreitol. Arg-tRNA synthetase was not reactivated immediately after the addition of dithiothreitol, but rather after the complete reactivation of Lys-tRNA synthetase. Leu-tRNA synthetase was inactivated to the largest extent and was irreversible. The initial lags in the inactivation of these synthetases may be due to the presence of dithiothreitol (1 raM) in the mix from the storage solution of the synthetase complex. Small but significant inactivation of Met-tRNA synthetase by ascorbate appears to follow a time-course of inactivation similar to that of Leu-tRNA synthetase. Ile-tRNA synthetase was minimally affected by oxidation or by the inactivation of the other synthetases.

Thermal inactivation of high-Mr aminoacyl-tRNA synthetases Thermal inactivation of proteins provides another approach for probing the effect of intermolecular interactions within the same multienzyme complex. However, the perturbation on the conformation of protein is rather general and nonspecific in comparison to that of oxidative modification. Kinetics of thermal inactivation of Arg-, Ile-, Leu-, Lys- and Met-tRNA synthetases in the purified 18 S high-M r complex are shown in Fig. 2b. The five different synthetase activities were inactivated at different rates. Met- and Leu-tRNA synthetases showed a transient increase in activity before inactivation began with a remarkable parallel in their time-courses. Arg- and Lys-tRNA synthetase activities were inactivated most rapidly with non-first-order kinetics. The inactivation kinetics of synthetases for Arg and Lys are almost identical, with residual activity at about 60% of initial activity. IIe-tRNA synthetase was not inactivated at all, despite the inactivation of other synthetases in the same complex. Similar thermal inactivation time-courses were obtained in the presence of bovine serum albumin at 45°C (data not shown).

The inset (Fig. 2b) shows the comparative thermal inactivation kinetics of 24 S and 6 S Lys-tRNA synthetase [13]. The 6 S enzyme was inactivated rapidly with a first-order kinetics, while the 24 S enzyme was clearly less thermolabile. The 24 S Lys-tRNA synthetase similar to 18 S Lys-tRNA synthetase also showed residual activity at about 60% of initial activity. Discussion Determination of Michaelis constants revealed no significant differences in most of the K,, values of unassociated and high-M r complex aminoacyl-tRNA synthetases. This observation is consistent with the finding that proteolytically dissociated Lys- and Met-tRNA synthetases purified from sheep liver exhibited the same K m values as those found in the multienzyme complex [21]. The K m values for rat liver Lys- and Met-tRNA synthetases in this communication are similar to those of the respective sheep liver enzymes [21], but quite different from those of the rat liver 12 S Lysand Arg-tRNA synthetase complex [24]. The Km values of Lys-tRNA synthetase in the 12 S complex are about 10-fold or higher than those obtained in this communication. The 12 S complex preparation contained Nonidet P-40, which has been reported to inhibit rat liver Arg-tRNA synthetase [25]. Functional independent of synthetases in the complex was noted previously by Mirande et al. [21] by comparing proteolytically cleaved Lys- and Met-tRNA synthetases with those in the synthetase complex from sheep liver. Our present analysis examining Lys-, Arg-, Leu-, Ile- and MettRNA synthetases dissociated from the complex by hydrophobic interaction chromatography further support the hypothesis that all synthetases aminoacylate independently in the synthetase complex. Employing thermal inactivation of synthetases, we examined possible interdependence of the thermal stability of aminoacyl-tRNA synthetases within the same complex. The time-courses of the thermal inactivation of different high-M r synthetases are more complex than the simple firstorder kinetics. The nature of the complexity is not understood, but they may reflect possible in-

325 termolecular stabilization among neighboring synthetases in the synthetase complex. The residual activities represent the complexed enzyme form which is more resistant to thermal inactivation than the free enzyme due to the intermolecular interactions. Studies of mutants of Chinese hamster ovary Leu-tRNA synthetase are consistent with possible stabilization of Leu-tRNA synthetase by complex formation [14]. Oxidative modification of proteins has been proposed as one of the cellular mechanisms for marking proteins destined for degradation [20]. Under appropriate conditions, the action of ascorbate resembles mixed function oxidation, specifically oxidizes an essential histidine residue and some as yet unidentified residues in glutamine synthetase [23] and inactivates the enzyme [22]. Demonstration of the selective inactivation of Leu-tRNA synthetase in the synthetase complex provides a unique opportunity for studying the turnover of large protein complexes through such mechanism, since the inactivation and the subsequent degradation of the different synthetases in the complex can be monitored separately. The rather specific irreversible inactivation of LeutRNA synthetase apparently did not affect other synthetase activities in the same complex. Inactivation of Lys- and Arg-tRNA synthetases can be reversed by dithiothreitol and most likely involves essential cysteine residues rather than histidine residues. Most soluble proteins as well as dissociated synthetases exhibited first-order kinetics in thermal inactivation or oxidative-modification inactivation. The non-first-order kinetics in these inactivation processes exhibited by enzymes in the synthetase complex, in particular the leveling off of inactivation at a certain level, are probably due to the intermolecular interaction in the supramolecular structure. Parallel time-courses in the inactivation processes of the component enzymes in a protein complex, especially concurrent inactivation and reactivation or vice versa, suggest coupled stability and lability of the component enzymes. Thus, coupled as well as non-first-order inactivation kinetics in turn suggest mutual stabilization and destabilization among neighboring enzymes in the synthetase complex through intermolecular interaction. In thermal inactivation, Leu- and Met-

tRNA synthetases, which clearly showed parallel activation and inactivation as well as non-firstorder kinetics, suggest their functional interaction in terms of thermal stability. Similarly, the parallel time-courses of inactivation by oxidative modification and subsequent reactivation by dithiothreitol of Lys- and Arg-tRNA synthetases suggest their mutual stabilization. It is intriguing that Lys- and Arg-tRNA synthetases showed remarkably parallel time-courses in both thermal and oxidative inactivations, which act through entirely different mechanisms. Thus, the stability of these two enzymes appears to be coupled, although Lys- and Arg-tRNA synthetases function independently in the multienzyme complex. Lys-tRNA synthetase can be completely dissociated from the synthetase complex and remains fully active [18], while dissociated Arg-tRNA synthetase becomes extremely unstable (Yang, unpublished results). It appears that stability of ArgtRNA synthetase depends appreciably on its association with fully active Lys-tRNA synthetase. This notion is strengthened substantially by the fact that both inactivation and subsequent reactivation of Arg-tRNA synthetase lagged behind those of Lys-tRNA synthetase during oxidative modification. Although free arginyl-tRNA synthetase has been isolated [26], it should be noted that it has a smaller subunit M r than that in the synthetase complex [8,9] and thus may be proteolytically cleaved or a different gene product. Studies of the dissociation behavior of particulate synthetases showed that Lys- and ArgtRNA synthetases and Leu- and Met-tRNA synthetases remain associated toward the end of the disassembly scheme [19]. Synthetase complexes consisting of Lys- and Arg-tRNA synthetases have been isolated [27] and subsequently purified [9]. These results suggest that Lys- and Arg-tRNA synthetases and possibly Leu- and Met-tRNA synthetases are relatively tightly associated, and suggest the physical proximity of these enzymes in the synthetase complex. Our present results provide a first piece of suggestive evidence for specific functional interaction between Lys- and Arg-tRNA synthetases and possibly between Leu- and MettRNA synthetases in the synthetase complex. Such specific functional interaction is in accord with the specific interaction among these synthetases de-

326

rived from the dissociation behavior of the synthetase complex [19]. In summary, different aminoacyl-tRNA synthetases in the synthetase complex catalyze various aminoacylation reactions independently. No appreciable effects on the Michaelis constants for amino acids, ATP or tRNA due to the association of synthetases were found. However, the stabilities of several synthetases in the synthetase complex appear to be interdependent; most notably, those of Lys- and Arg-tRNA synthetases. Such effect on the stability of these enzymes provides a new approach to elucidating the structural and functional interactions among the component enzymes in multienzyme complexes. Other aspects of possible functional significance of the association of the aminoacyl-tRNA synthetases remain to be explored, such as compartmentalization of the protein biosynthetic machinery and regulation of other cellular processes such as the biosynthesis of diadenosine tetraphosphate.

Acknowledgements One of us (D.Y.) gratefully acknowledges the stimulating discussions with Drs. P. Boon Chock, Charles Huang, Rodney L. Levine and Earl R. Stadtman on the experiment of oxidative modification, which was carried out while on sabbatical in their laboratory. This investigation was supported by grants from NSF (81-10818) and NIH (GM 25848).

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