Subcellular distribution and properties of rabbit liver aminoacyl-tRNA synthetases under myocardial ischemia

Subcellular distribution of aminoacyl-tRNA synthetase activities has been studied in normal rabbit liver and under experimental myocardial ischemia (EMI). An increase in the activity of a number of aminoacyl-tRNA synthetases in postmitochondrial and postribosomal supernatants from rabbit liver has been determined 12 hr after EMI. Gel chromatography of the postribosomal supernatant on Sepharose 6B shows that aminoacyl-tRNA synthetase activities are distributed among the fractions with M_r 1.82×10⁶, 0.84×10⁶ (high-M_r aminoacyl-tRNA synthetase complexes) and 0.12–0.35×10⁶. In the case of EMI aminoacyl-tRNA synthetase activities are partly redistributed from the 1.82×10⁶ complex into the 0.84×10⁶ complex. The catalytic properties of both free and complex leucyl-tRNA synthetases have been compared. K_M for all the substrates are the values of the same order in norm and under EMI. A decrease in some aminoacyl-tRNA synthetase activities associated with polyribosomes has been observed 12 hr after EMI. The interaction of aminoacyl-tRNA synthetases with polyribosomes stimulates the catalytic activity of some enzymes and protects them from heat inactivationin vitro. It is assumed that the changes in association of aminoacyl-tRNA synthetases with high-M_r complexes and compartmentalization of these enzymes on polyribosomes may be related to the alteration of protein biosynthesis under myocardial ischemia.     

A role for lipids in the functional and structural properties of the rat liver aminoacyl-tRNA synthetase complex

The high molecular weight aminoacyl-tRNA synthetase complexes found in extracts of many eukaryotic cells often contain lipids and other non-protein components. Since hydrophobic interactions play an important role in maintaining synthetases in the complex, it has been suggested that the lipids present may also participate in its functional and structural integrity. In order to learn more about the role of lipids in the complex, we have compared the properties of the normal complex to one which has been delipidated by treatment with Triton X-114. Delipidation does not affect the size or activity of the aminoacyl-tRNA synthetase complex, but a variety of functional and structural properties of individual synthetases in the complex are altered dramatically. These include sensitivity to salts plus detergents, temperature inactivation, hydrophobicity, and sensitivity to protease digestion. In the latter case, removal of lipids also affects the low molecular weight products released by protease digestion. Purification of the synthetase complex by various chromatographic procedures can remove the lipids and lead to a structure that behaves like the delipidated complex prepared by detergent treatment. The significance of these findings for the intracellular location of aminoacyl-tRNA synthetases and for the study of purified complexes are discussed.     

Isolation and electron microscopic characterization of the high molecular mass aminoacyl-tRNA synthetase complex from murine erythroleukemia cells

A high molecular mass aminoacyl-tRNA synthetase complex has been isolated from a murine erythroleukemia cell line. This multienzyme complex contains activities for the arginyl-, aspartyl-, glutamyl-, glutaminyl-, isoleucyl,- leucyl-, lysyl-, methionyl-, and prolyl-tRNA synthetases. This enzyme composition, the polypeptide pattern observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the relative stoichiometry of the component polypeptides are characteristic of high molecular mass complexes of aminoacyl-tRNA synthetases isolated from a variety of mammalian tissues and cell types. Negatively stained preparations of native complex and of glutaraldehyde-treated material have been examined by electron microscopy. In both cases, a distinctive particle is observed which appears in several orientations. The most common views are of two different projections of a squarish particle that measures approximately 27 x 27 nm. Other commonly observed views are of a "U" shape, a rectangle, and a triangle. All of these views are seen in both gradient-purified samples and those prepared directly from material as isolated. These data are consistent with a model for the multienzyme aminoacyl-tRNA synthetase complex as a "cup" or elongated U structure. These studies demonstrate that the high molecular mass complex of eukaryotic aminoacyl-tRNA synthetases does have a coherent structure that can be visualized by electron microscopy.     

Interaction of brain transferase I with guanosine nucleotides and aminoacyl-tRNA

Brain transferase I has been purified, and its interactions with GTP and aminoacyl-tRNA have been studied. The data suggest that a transferase I-GTP complex can be formed which interacts with aminoacyl-tRNA to yield an aminoacyl-tRNA-transferase I-GTP complex.     

Seven mammalian aminoacyl-tRNA synthetases associated within the same complex are functionally independent

A heterotypic multienzyme complex from sheep liver containing seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, glutamine, glutamic acid, lysine and arginine was subjected to kinetic analyses to examine the possibility that association of these enzymes may impart kinetic properties which differ from those of their unassociated counterparts. The evidence obtained by two different approaches leads to the conclusion that the associated enzymes are functionally independent. Firstly, the kinetic constants of the methionyl-tRNA and lysyl-tRNA synthetase components of the complex do not differ significantly from those of their unassociated counterparts obtained after controlled proteolysis of the complex. Secondly, the methionyl-tRNA synthetase component of the complex displays identical kinetic constants, whether assayed in the presence of [14C]methionine, ATP and highly enriched tRNAMet alone, or in the additional presence of the substrates required for unlabeled aminoacyl-tRNA formation by each of the other six enzymes. Similarly, the initial rates of [14C]aminoacyl-tRNA formation catalyzed by any of the six other enzymes was unaffected by the concomitant functioning of the other aminoacyl-tRNA synthetases. The sedimentation behaviour of the aminoacyl-tRNA synthetase components of the complex under conditions prevailing in the tRNA aminoacylation assay indicates that they remain associated under these conditions. The implications of these findings on the structural organization of the enzymes within the complex are discussed.     

Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. The role of guanosine triphosphate

1. Transferase I from rat liver binds relatively low quantities of GTP when incubated with this nucleotide in the absence of aminoacyl-tRNA. 2. Transferase I reacts with both aminoacyl-tRNA and GTP to form a relatively stable complex that is retained on cellulose nitrate filters. The ternary complex transferase I-aminoacyl-tRNA-GTP is also formed when the transferase I-aminoacyl-tRNA complex is incubated with GTP or during the incubation of the transferase I-GTP complex with aminoacyl-tRNA. Synthesis of this complex does not require the presence of Mg(2+). 3. In the presence of Mg(2+) the ternary complex becomes readily bound to ribosomes without requirements for any other cofactors. 4. An extensive cleavage of GTP takes place when aminoacyl-tRNA becomes bound to ribosomes. 5. The low interdependence of reactions leading to the formation of transferase I complexes with aminoacyl-tRNA and GTP indicates that the mechanisms of the binding reaction in mammalian systems may be different from those in bacterial cells.     

Coordination of Mn++ ions at contact sites between tRNA and aminoacyl-tRNA synthetase.

By means of PMR and ESR study the shielding of Mn⁺⁺ ions by aminoacyl-tRNA synthetase has been detected in the aminoacyl-tRNA synthetase - tRNA complex at pH 7.5. At pH 6 this effect was not observed. We propose that ions interact with certain aminoacyl-tRNA synthetase groups protonated when passing to slightly acid pH. The role of Mn⁺⁺ and Mg⁺⁺ ions in the formation of a functionally active complex tRNA-aminoacyl-tRNA synthetase is discussed.     

Role of aminoacyl-tRNA-synthetases in changes in the rate of protein biosynthesis in the rabbit liver during myocardial ischemia

Composition of high-molecular-weight aminoacyl-tRNA synthetases complexes from rabbit liver both in norm and after 12 h experimental myocardial ischemia (EMI) has been investigated. Partial redistribution of aminoacyl-tRNA synthetases activity from 1820 kD complex into 840 kD complex was observed in case of EMI which resulted in changes of protein biosynthesis rate in cell-free system.     

Synthesis of diadenosine 5',5' -P1,P4-tetraphosphate by lysyl-tRNA synthetase and a multienzyme complex of aminoacyl-tRNA synthetases from rat liver

An 18 S multienzyme complex of aminoacyl-tRNA synthetases is found to be active in the synthesis of diadenosine-5',5'-P1,P4-tetraphosphate (AppppA). Most of the activity is attributed to lysyl-tRNA synthetase in the complex. Free lysyl-tRNA synthetase dissociated from the synthetase complex is about 6-fold more active than the complex in AppppA synthesis, while their apparent Michaelis constants for ATP and lysine are similar. AMP, which reportedly activates AppppA synthesis (Hilderman, R.H. (1983) Biochemistry 22, 4353-4357), has no effect on AppppA synthesis. The higher activity of free Lys-tRNA synthetase is in part due to the higher stimulation of AppppA synthesis by Zn2+. These results suggest that association of aminoacyl-tRNA synthetases may affect AppppA synthesis.     

Functional Compartmentalization of the Translation Apparatus and Channeling of tRNA/Aminoacyl-tRNA in Cells of Higher Eukaryotes

Literature data and authors' results on the structural and functional organization of the translation apparatus in higher eukaryotes are considered. Proofs are presented of the channeling of tRNA/aminoacyl-tRNA in the course of eukaryotic protein synthesis. The concept of the shuttle role of eEF1A is grounded; the factor, being in a GTP-bound form, delivers aminoacyl-tRNA to the ribosome and then, in the having undergone to a GDP -form after hydrolysis of GTP on the ribosome, forms a complex with the deacylated tRNA and delivers it to the aminoacyl-tRNA synthetase. The notion of a translational compartment is defined.     

Effects of ricin on the ribosomal sites involved in the interaction of the elongation factors.

The effects of ricin on the different steps of the elongation cycle of protein synthesis in a rabbit reticulocyte cell-free system are studied in this paper. The toxin most probably acts by catalytically inactivating the ribosomes, since a single molecule of the toxin can inactivate 300 ribosomes for poly(U)-directed phenylalanine incorporation. The effect of the toxin on the ribosome is irreversible. Ricin specifically inhibits elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes but has no effect on the non-enzymic binding of aminoacyl-tRNA. Ricin also inhibits formation of the complex elongation-factor-2 - ribosome - nucleotide with GTP, GDP or GMP-P(CH2)P. However, the toxin has no effect on translocation. These apparently conflicting results are discussed in this study.     

High-molecular-weight forms of aminoacyl-tRNA synthetases and tRNA modification enzymes in Escherichia coli.

The presence of high-molecular-weight complexes of aminoacyl-tRNA synthetases in Escherichia coli has been reported (C. L. Harris, J. Bacteriol. 169:2718-2723, 1987). In the current study, Bio-Gel A-5M gel chromatography of 105,000 x g supernatant preparations from E. coli Q13 indicated high molecular weights for both tRNA methylase (300,000) and tRNA sulfurtransferase (450,000). These tRNA modification enzymes did not appear to exist in the same multienzymic complex. On the other hand, 4-thiouridine sulfurtransferase eluted with aminoacyl-tRNA synthetase activity on Bio-Gel A-5M, and both of these activities were cosedimented after further centrifugation of cell supernatants at 160,000 x g for 18 h. Despite this evidence for association of the sulfurtransferase with the synthetase complex, isoleucyl-tRNA synthetase and tRNA sulfurtransferase were totally resolved from each other by DEAE-Sephacel chromatography. Subsequent gel chromatography showed little change in their elution positions on agarose. Hence, either nonspecific aggregation occurred here, or the modification enzymes studied are not members of the aminoacyl-tRNA synthetase complex in E. coli. These findings do suggest that some bacterial tRNA modification enzymes are present in multiprotein complexes of high molecular weight.     

Functional compartmentation of the translation apparatus and channeling of tRNA/aminoacyl-tRNA in cells of higher eukaryotes

Literature data and authors' results in the field of structure-function organization of the translation apparatus in higher eukaryotes are considered. Proof is presented of the channeling of tRNA/aminoacyl-tRNA in the course of eukaryotic protein synthesis. The concept of the shuttle role of elongation factor eEF1A is grounded; the factor, being in a GTP-bound form, delivers aminoacyl-tRNA to the ribosome and then, in the GDP form after hydrolysis of GTP on the ribosome, forms a complex with the deacylated tRNA and delivers it to the aminoacyl-tRNA synthetase. The notion of translational compartment is defined.     

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).     

Structural analysis of the high molecular mass aminoacyl-tRNA synthetase complex. Effects of neutral salts and detergents.

The effects of a variety of detergents and neutral salts on the structure of the eukaryotic high molecular mass aminoacyl-tRNA synthetase complex have been directly determined by observing alterations in the composition, sedimentation behavior, and electron microscopic appearance of the rabbit reticulocyte complex. The intact complex is shown to exhibit the enzymatic activities, polypeptide composition, relative stoichiometry, and morphological features that are characteristic of this eukaryotic multienzyme particle. The structure of the high molecular mass aminoacyl-tRNA synthetase complex is seen to be resistant to both ionic and nonionic detergents. However, both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and deoxycholate induce formation of large protein aggregates. In contrast, the chaotropic salts LiCl and NaSCN both selectively remove individual polypeptides from the high molecular mass aminoacyl-tRNA synthetase complex and promote formation of specific particulate subcomplexes which have distinct sizes, polypeptide compositions, and structural features. These data support the view that many of the protein interactions within the high molecular mass amino-acyl-tRNA synthetase complex are hydrophobic in nature. This study also provides direct evidence that the complex contains a core of tightly interacting synthetases onto which the remaining polypeptides are arrayed. The structural alterations observed here may account for the ability of these reagents to markedly inhibit several enzymatic activities within the complex.     

Alteration of aminoacyl-tRNA synthetase activities by phosphorylation with casein kinase I

The phosphorylation of a highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes by the cyclic nucleotide-independent protein kinase, casein kinase I, has been examined, and the effects of phosphorylation on the synthetase activities were determined. The synthetase complex, purified as described (Kellermann, O., Tonetti, H., Brevet, A., Mirande, M., Pailliez, J.-P., and Waller, J.-P. (1982) J. Biol. Chem. 257, 11041-11048), contains seven aminoacyl-tRNA synthetases and four unidentified proteins and is free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP results in the phosphorylation of four synthetases, namely, glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I alters binding of the aminoacyl-tRNA synthetase complex to tRNA-Sepharose. The phosphorylated synthetase complex elutes from tRNA-Sepharose at 190 mM NaCl, while the nonphosphorylated complex elutes at 275 mM NaCl. Phosphorylation by casein kinase I results in a significant inhibition of aminoacylation by the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases; the activities of the nonphosphorylated synthetases remain unchanged. These data indicate that phosphorylation of aminoacyl-tRNA synthetases in the high molecular weight complex alters the activities of these enzymes. One of the unidentified proteins present in the complex (Mr 37,000) is also highly phosphorylated by casein kinase I. From a comparison of the properties and phosphopeptide pattern of this protein with that of casein kinase I, it appears that the Mr 37,000 protein in the synthetase complex is an inactive form of casein kinase I. This observation provides further evidence for a physiological role for casein kinase I in regulating synthetase activities.     

A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome.

Elongation factor Tu (EF-Tu).GTP has the primary function of promoting the efficient and correct interaction of aminoacyl-tRNA with the ribosome. Very little is known about the elements in EF-Tu involved in this interaction. We describe a mutant form of EF-Tu, isolated in Salmonella typhimurium, that causes a severe defect in the interaction of the ternary complex with the ribosome. The mutation causes the substitution of Val for Gly-280 in domain II of EF-Tu. The in vivo growth and translation phenotypes of strains harboring this mutation are indistinguishable from those of strains in which the same tuf gene is insertionally inactivated. Viable cells are not obtained when the other tuf gene is inactivated, showing that the mutant EF-Tu alone cannot support cell growth. We have confirmed, by partial protein sequencing, that the mutant EF-Tu is present in the cells. In vitro analysis of the natural mixture of wild-type and mutant EF-Tu allows us to identify the major defect of this mutant. Our data shows that the EF-Tu is homogeneous and competent with respect to guanine nucleotide binding and exchange, stimulation of nucleotide exchange by EF-Ts, and ternary complex formation with aminoacyl-tRNA. However various measures of translational efficiency show a significant reduction, which is associated with a defective interaction between the ribosome and the mutant EF-Tu.GTP.aminoacyl-tRNA complex. In addition, the antibiotic kirromycin, which blocks translation by binding EF-Tu on the ribosome, fails to do so with this mutant EF-Tu, although it does form a complex with EF-Tu. Our results suggest that this region of domain II in EF-Tu has an important function and influences the binding of the ternary complex to the codon-programmed ribosome during protein synthesis. Models involving either a direct or an indirect effect of the mutation are discussed.     

Euglena gracilis chloroplast elongation factor Tu. Interaction with guanine nucleotides and aminoacyl-tRNA

The interaction of the chloroplast elongation factor Tu (EF-Tuchl) from Euglena gracilis with guanine nucleotides and aminoacyl-tRNA has been investigated. The apparent dissociation constant at 37 degrees C for the EF-Tuchl X GDP complex is about 3 X 10(-7) M and for the EF-Tuchl X GTP complex, it is about 1 order of magnitude higher. The sulfhydryl modifying reagent N-ethylmaleimide severely inhibits the polymerization activity of Euglena EF-Tuchl. In the presence of N-ethylmaleimide, the dissociation constant for the modified EF-Tuchl X GDP complex is increased by an order of magnitude. Conversely, both GDP and GTP protect EF-Tuchl from the modification. The polymerization activity of EF-Tuchl is also sensitive to the antibiotic kirromycin. In the presence of kirromycin, the apparent dissociation constant for the EF-Tuchl X GTP complex is lowered 10-fold. The interaction of aminoacyl-tRNA with EF-Tuchl was investigated by examining the ability of EF-Tuchl to prevent the spontaneous hydrolysis of Phe-tRNA and by gel filtration chromatography. The binding of aminoacyl-tRNA to EF-Tuchl occurs only in the presence of GTP indicating the formation of the ternary complex EF-Tuchl X GTP X Phe-tRNA. The effect of kirromycin on the interaction was also investigated. In the presence of kirromycin, no interaction between EF-Tuchl and Phe-tRNA is observed, even in the presence of GTP.     

The influence of different modifications of elongation factor Tu from Escherichia coli on ternary complex formation investigated by fluorescence spectroscopy.

A fluorescence titration assay was used to detect the effects of various modifications of E.coli elongation factor Tu on the formation of the ternary complex with aminoacyl-tRNAs. The treatment of EF-Tu.GDP with TPCK, an analogue of the 3'terminus of aminoacyl-tRNA, was found to have no influence on the conversion of EF-Tu.GDP to 'active' EF-Tu.GTP, but does decrease the affinity of the activated protein for yeast aminoacyl-tRNA by more than three orders of magnitude. Modification of the elongation factor by limited cleavage with trypsin, leading to the excision of amino acid residues 45-58, has only a minor influence on ternary complex formation. The equilibrium dissociation constant of the ternary complex with this trypsin-treated EF-Tu.GTP and E.coli Phe-tRNA(Phe) is only one order of magnitude higher than that of the ternary complex with native EF-Tu. Mutations in the amino acid residues 222 and 375 of EF-Tu also have little effect on ternary complex formation. Compared with TPCK-treated EF-Tu, the affinities of the two mutant species, designated EF-tuAR and EF-TuBO respectively, for [AEDANS-s2C]Tyr-tRNA(Tyr) are only slightly reduced and in the same range as trypsin-cleaved EF-Tu.     

A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA

Codon recognition by aminoacyl-tRNA on the ribosome triggers a process leading to GTP hydrolysis by elongation factor Tu (EF-Tu) and release of aminoacyl-tRNA into the A site of the ribosome. The nature of this signal is largely unknown. Here, we present genetic evidence that a specific set of direct interactions between ribosomal protein S12 and aminoacyl-tRNA, together with contacts between S12 and 16S rRNA, provide a pathway for the signaling of codon recognition to EF-Tu. Three novel amino acid substitutions, H76R, R37C, and K53E in Thermus thermophilus ribosomal protein S12, confer resistance to streptomycin. The streptomycin-resistance phenotypes of H76R, R37C, and K53E are all abolished by the mutation A375T in EF-Tu. A375T confers resistance to kirromycin, an antibiotic freezing EF-Tu in a GTPase activated state. H76 contacts aminoacyl-tRNA in ternary complex with EF-Tu and GTP, while R37 and K53 are involved in the conformational transition of the 30S subunit occurring upon codon recognition. We propose that codon recognition and domain closure of the 30S subunit are signaled through aminoacyl-tRNA to EF-Tu via these S12 residues.