Functional Interaction of Mammalian Valyl-tRNA Synthetase with Elongation Factor EF-1α in the Complex with EF-1H

Abstract

In mammalian cells valyl-tRNA synthetase (ValRS) forms a high M r complex with the four subunits of elongation factor EF-1H. The β, γ, and δ subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1α could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1α and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAValsynthesis catalyzed by the valyl-tRNA synthetase component of the ValRS·EF-1H complex. This effect is not observed in the presence of EF-1α and GDP or EF-Tu·GTP and requires association of valyl-tRNA synthetase within the ValRS·EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1α catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1α·GTP·Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1α·GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells. In mammalian cells valyl-tRNA synthetase (ValRS) forms a high M r complex with the four subunits of elongation factor EF-1H. The β, γ, and δ subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1α could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1α and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAValsynthesis catalyzed by the valyl-tRNA synthetase component of the ValRS·EF-1H complex. This effect is not observed in the presence of EF-1α and GDP or EF-Tu·GTP and requires association of valyl-tRNA synthetase within the ValRS·EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1α catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1α·GTP·Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1α·GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells. Aminoacyl-tRNA is the donor of amino acid in ribosomal protein synthesis. The tRNA molecule is aminoacylated with the corresponding amino acid by an aminoacyl-tRNA synthetase, the aminoacyl-tRNA is converted to a ternary complex with elongation factor 1α, to give the immediate precursor of amino acid for protein synthesis: EF-1α·GTP·aminoacyl-tRNA. Several lines of evidence have suggested that in mammalian cells the translational apparatus is highly organized. In particular, association of the protein vectors of tRNA, aminoacyl-tRNA synthetases and elongation factors, with the cytoskeletal framework has been reported (1Dang C.V. Yang D.C.H. Pollard T.D. J. Cell Biol. 1983; 96: 1138-1147Crossref PubMed Scopus (78) Google Scholar, 2Mirande M. Le Corre D. Louvard D. Reggio H. Pailliez J.P. Waller J.P. Exp. Cell Res. 1985; 156: 91-102Crossref PubMed Scopus (75) Google Scholar, 3Sanders J. Brandsma M. Janssen G.M.C. Dijk J. Möller W. J. Cell Sci. 1996; 109: 1113-1117Crossref PubMed Google Scholar) and colocalization of these components described (4Barbarese E. Koppel D.E. Deutscher M.P. Smith C.L. Ainger K. Morgan F. Carson J.H. J. Cell Sci. 1995; 108: 2781-2790PubMed Google Scholar). The isolation and characterization of supramolecular assemblies of aminoacyl-tRNA synthetases and elongation factors (5Mirande M. Prog. Nucleic Acid Res. Mol. Biol. 1991; 40: 95-142Crossref PubMed Scopus (214) Google Scholar, 6Kisselev L.L. Wolfson A.D. Prog. Nucleic Acid Res. Mol. Biol. 1994; 48: 83-142Crossref PubMed Scopus (89) Google Scholar, 7Yang D.C.H. Curr. Top. Cell. Regul. 1996; 34: 101-136Crossref PubMed Scopus (50) Google Scholar) have provided structural evidence for the subcellular organization of the protein synthesis machinery. The existence of a channeled tRNA cycle during mammalian protein synthesis provided functional evidence for cellular compartmentalization of translation (8Negrutskii B.S. Deutscher M.P. Proc. Natl Acad. Sci. U. S. A. 1991; 88: 4991-4995Crossref PubMed Scopus (157) Google Scholar, 9Negrutskii B.S. Stapulionis R. Deutscher M.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 964-968Crossref PubMed Scopus (114) Google Scholar, 10Stapulionis R. Deutscher M.P. Proc. Natl Acad. Sci. U. S. A. 1995; 92: 7158-7161Crossref PubMed Scopus (143) Google Scholar). According to the proposed channeling scheme, aminoacyl-tRNAs are vectorially transferred from the aminoacyl-tRNA synthetases to the ribosomes as ternary complexes EF-1α·GTP·aminoacyl-tRNA (8Negrutskii B.S. Deutscher M.P. Proc. Natl Acad. Sci. U. S. A. 1991; 88: 4991-4995Crossref PubMed Scopus (157) Google Scholar, 10Stapulionis R. Deutscher M.P. Proc. Natl Acad. Sci. U. S. A. 1995; 92: 7158-7161Crossref PubMed Scopus (143) Google Scholar). Moreover, the GDP form of EF-1α could be involved in the capture of deacylated tRNA at the exit site of the ribosome and its delivery to the synthetase (11Petrushenko Z.M. Negrutskii B.S. Ladokhin A.S. Budkevich T.V. Shalak V.F. El'skaya A.V. FEBS Lett. 1997; 407: 13-17Crossref PubMed Scopus (28) Google Scholar). Channeling, or direct transfer of metabolites from one enzyme to another in a metabolic pathway, is believed to increase significantly the efficiency of the overall reaction (12Srere P.A. Annu. Rev. Biochem. 1987; 56: 89-124Crossref PubMed Scopus (805) Google Scholar). For sequential metabolic enzymes, the stimulation of activity of the first enzyme induced by a protein-protein interaction with the second enzyme provides a structural basis for a channeling mechanism. As far as the protein biosynthesis machinery is concerned, the possible regulation of aspartyl- (13Reed V.S. Wastney M.E. Yang D.C.H. J. Biol. Chem. 1994; 269: 32932-32936Abstract Full Text PDF PubMed Google Scholar) and phenylalanyl- (14Negrutskii B.S. Budkevich T.V. Shalak V.F. Turkovskaya G.V. El'skaya A.V. FEBS Lett. 1996; 382: 18-20Crossref PubMed Scopus (36) Google Scholar) tRNA synthetase activities by elongation factor EF-1α has been reported. In both cases, no stable protein-protein interaction between the synthetase and the elongation factor was detected. In connection with tRNA channeling from aminoacyl-tRNA synthetase to EF-1α, two enzymes ensuring consecutive steps of the tRNA cycle, the multienzyme complex containing valyl-tRNA synthetase and EF-1H deserves special mention. The only stable macromolecular assemblage that involves an aminoacyl-tRNA synthetase and elongation factor 1α is the ValRS 1The abbreviations ValRSvalyl-tRNA synthetaseDTE1,4-dithioerythritolGMP-PNPβ;γ-imidoguanosine 5′-triphosphate 1The abbreviations ValRSvalyl-tRNA synthetaseDTE1,4-dithioerythritolGMP-PNPβ;γ-imidoguanosine 5′-triphosphate·EF-1H complex (15Bec G. Kerjan P. Zha X.D. Waller J.P. J. Biol. Chem. 1989; 264: 21131-21137Abstract Full Text PDF PubMed Google Scholar,16Motorin Y.A. Wolfson A.D. Löhr D. Orlovsky A.F. Gladilin K.L. Eur. J. Biochem. 1991; 201: 325-331Crossref PubMed Scopus (25) Google Scholar). EF-1H, the “heavy” form of the translation elongation factor 1, is a pentameric complex of the four subunits α, β, γ, and δ in molar ratio 2:1:1:1 (17Janssen G.M.C. van Damme H.T.F. Kriek J. Amons R. Möller W. J. Biol. Chem. 1994; 269: 31410-31417Abstract Full Text PDF PubMed Google Scholar). EF-1α forms a ternary complex with aminoacyl-tRNA and GTP to give the active species of amino acid for protein synthesis and delivers aminoacyl-tRNA to the A site of the ribosome. The EF-1βγδ subunits contribute the guanine nucleotide exchange activity to regenerate EF-1α·GTP from EF-1α·GDP. The finding that in mammalian cells valyl-tRNA synthetase is exclusively found as a complex with EF-1H has suggested that this association might contribute an essential function in vivo. Here we show that EF-1α controls the aminoacylation reaction catalyzed by ValRS. Our results demonstrate that association of ValRS with EF-1H is absolutely required for the stimulation in trans by EF-1α. The ValRS·EF-1H complex provides a structural basis for the functional interaction between ValRS and EF-1α. The coupling of these two consecutive reactions during protein biosynthesis could have a regulatory function. valyl-tRNA synthetase 1,4-dithioerythritol β;γ-imidoguanosine 5′-triphosphate valyl-tRNA synthetase 1,4-dithioerythritol β;γ-imidoguanosine 5′-triphosphate The ValRS·EF-1H complex was isolated from rabbit liver according to a procedure adapted from that used to isolate the multisynthetase complex (18Mirande M. Le Corre D. Waller J.P. Eur. J. Biochem. 1985; 147: 281-289Crossref PubMed Scopus (81) Google Scholar). Briefly, livers (1 kg) from 13 rabbits were homogenized in three portions (w/v; total volume 2 liters) of extraction buffer (50 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 0.1 mm EDTA, 10% glycerol, 1 mm DTE (1,4-dithioerythritol)) containing 1 mm diisopropyl fluorophosphate. Postmitochondrial supernatant was adjusted to 2% polyethylene glycol-6000 by addition of a 50% stock solution in extraction buffer and stirred for 30 min at 4 °C. After centrifugation at 10,000 × g for 30 min, the supernatant was recovered, adjusted to 5% polyethylene glycol-6000, and stirred for 30 min at 4 °C. After centrifugation, the precipitate was dissolved in 200 ml of 25 mm potassium phosphate (pH 7.6), 5% glycerol, and 10 mm2-mercaptoethanol. The homogeneous solution was applied to a Bio-Gel A-5m column (Bio-Rad; 90 × 910 mm) equilibrated with the same buffer containing 10% glycerol. Fractions containing valyl-tRNA synthetase activity were combined and loaded on a tRNA-Sepharose column (50 × 315 mm) equilibrated with the same buffer. The enzyme was eluted by a linear gradient of 25–400 mm potassium phosphate buffer (pH 7.6). Fractions with valyl-tRNA synthetase activity were pooled and applied to a Source 15Q column (Amersham Pharmacia Biotech; 20 × 95 mm) equilibrated in 25 mmTris-HCl (pH 7.5), 50 mm KCl, 10% glycerol, and 10 mm 2-mercaptoethanol and developed with a 50–1000 mm KCl gradient in the same buffer. Fractions with ValRS activity were pooled, concentrated by vacuum dialysis, and stored at −20 °C after dialysis against 25 mm potassium phosphate (pH 7.6), 50% glycerol, and 2 mm DTE. Dissociation of the ValRS·EF-1H complex and isolation of its ValRS, EF-1δ, and EF-1βγ subunits in their native state was conducted in the presence of 0.5 m NaSCN as reported previously (19Bec G. Waller J.P. J. Biol. Chem. 1989; 264: 21138-21143Abstract Full Text PDF PubMed Google Scholar). The free truncated ValRS species deprived of its NH2-terminal extension was obtained by controlled elastase treatment, as described (20Bec G. Kerjan P. Waller J.P. J. Biol. Chem. 1994; 269: 2086-2092Abstract Full Text PDF PubMed Google Scholar). The free form of elongation factor 1α was purified to homogeneity from rabbit liver as described in (14Negrutskii B.S. Budkevich T.V. Shalak V.F. Turkovskaya G.V. El'skaya A.V. FEBS Lett. 1996; 382: 18-20Crossref PubMed Scopus (36) Google Scholar) and stored at −80 °C in 25 mm Tris-HCl (pH 7.5), 25 mm NH4Cl, 2 mm MgCl2, 2 mm DTE, and 25% glycerol. ValRS from Saccharomyces cerevisiae, phenylalanyl-tRNA synthetase, and the multisynthetase complex from rabbit liver were purified as described previously (14Negrutskii B.S. Budkevich T.V. Shalak V.F. Turkovskaya G.V. El'skaya A.V. FEBS Lett. 1996; 382: 18-20Crossref PubMed Scopus (36) Google Scholar, 18Mirande M. Le Corre D. Waller J.P. Eur. J. Biochem. 1985; 147: 281-289Crossref PubMed Scopus (81) Google Scholar, 19Bec G. Waller J.P. J. Biol. Chem. 1989; 264: 21138-21143Abstract Full Text PDF PubMed Google Scholar). Homogeneous EF-Tu fromEscherichia coli was generously provided by Dr. Andrea Parmeggiani (Ecole Polytechnique, Palaiseau, France). Initial rates of tRNA aminoacylation were measured at 25 °C in 0.1 ml of 20 mm imidazole-HCl buffer (pH 7.5), 3 mm KCl, 15% glycerol, 1 mg/ml bovine serum albumin, 0.5 mm DTE, 5 mmMgCl2, 3 mm ATP, 60 μm[14C]valine (NEN Life Science Products, 50 Ci/mol), and saturating amounts (5–10 μm) of partially purified beef liver tRNA (valine acceptance of 325 pmol/A 260). Where indicated, the incubation mixture also contained GDP, GTP, or with or EF-1α. amounts of ValRS were after in 20 mmTris-HCl (pH 7.5), 10% glycerol, mm DTE, and 4 mg/ml bovine serum and were conducted in The reaction mixture was at 25 °C for 2 min and the aminoacylation reaction was by addition of was conducted at 25 °C. were at and in 5% tRNA was on were and in of activity is the of enzyme one of at 25 °C. of synthesis by ValRS from the ValRS·EF-1H complex in the presence or in the of an excess of free EF-1α were obtained in the presence of GTP in the incubation m were by of the to the the For the activity of EF-1α, the guanine nucleotide exchange was as described previously (20Bec G. Kerjan P. Waller J.P. J. Biol. Chem. 1994; 269: 2086-2092Abstract Full Text PDF PubMed Google Scholar). The complex was incubation of EF-1α with 4 (Amersham Pharmacia Biotech; in of mm Tris-HCl (pH containing 0.5 mm DTE, mm mm NH4Cl, 1 mg/ml bovine serum albumin, and for 5 min at °C. The reaction mixture was on and by addition of of exchange buffer mm Tris-HCl (pH 7.5), 10 mm 50 mm NH4Cl, 0.1 mg/ml bovine serum The exchange reaction was conducted at °C after addition of exchange buffer containing nucleotide and exchange were at and were three with 1 ml of buffer mm Tris-HCl (pH 7.5), 10 mm MgCl2, mm NH4Cl, 0.1 mg/ml bovine serum and in the GTP form of purified EF-Tu was with GTP in an incubation mixture containing 25 mm Tris-HCl (pH 7.5), 50 mm NH4Cl, 10 mm MgCl2, 1 mm DTE, 0.5 mm EDTA, in the presence of and (1 mm) to of was conducted at 30 °C for min, and the EF-Tu·GTP was used suggested that tRNA aminoacylation catalyzed by ValRS is of its association with EF-1α. the activity of the in the aminoacylation was not its from the EF-1H complex (15Bec G. Kerjan P. Zha X.D. Waller J.P. J. Biol. Chem. 1989; 264: 21131-21137Abstract Full Text PDF PubMed Google Scholar, 19Bec G. Waller J.P. J. Biol. Chem. 1989; 264: 21138-21143Abstract Full Text PDF PubMed Google Scholar). the and subunits are tightly to ValRS, the EF-1α be from the ValRS·EF-1H complex J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). This finding suggested that the ternary complex EF-1α·GTP·Val-tRNAVal is from the ValRS·EF-1H complex of a aminoacylation We that of by ValRS is controlled by EF-1α, this effect be only in the presence of an excess of free EF-1α. ValRS and EF-1α activities are as tRNA is channeled from the synthetase to the elongation addition in the reaction mixture of free EF-1α in that could be its EF-1α·GTP species by the β, γ, and δ subunits of EF-1H in the presence of GTP, in an of EF-1α is to a ternary complex with the aminoacylated tRNA, this stimulation we an aminoacylation to a effect of EF-1α·GTP on the efficiency catalyzed by the ValRS component of the ValRS·EF-1H complex. In the procedure used in study, the reaction mixture containing the ValRS·EF-1H complex and free EF-1α in deprived of tRNA, was 2 min at 25 °C the aminoacylation reaction was by addition of the tRNA This was to a in the described EF-1H is with EF-1α·GTP during this amounts of the ValRS·EF-1H complex is in the presence of an excess of free EF-1α and saturating amounts of the for the aminoacylation in the presence of GTP to the active species the of valyl-tRNAValsynthesis was 2-fold from to 1 GTP no effect on the of This aminoacylation was observed during the first of the the free form of EF-1α is in As in 1 with a of the ValRS·EF-1H complex of free EF-1α is the of is The in the presence of EF-1α·GTP of is 2-fold that observed in the of EF-1α After the reaction for min, of corresponding to the of EF-1α in the incubation mixture (50 the of aminoacylation to its in the of EF-1α. As in the of 1 in the presence of GTP the of the stimulation and the of synthesis are of the of free EF-1α in the incubation The GTP form of elongation factor EF-1α, and its to contribute a ternary complex is absolutely required to the aminoacylation activity of ValRS. in the presence of GDP of GTP, the of of by the ValRS·EF-1H complex is not by the presence of a excess of free EF-1α The GTP a stimulation of ValRS activity in the presence of EF-1α, to a as with GTP This efficiency could be to a of as with the to a of of from the ValRS·EF-1H as with the ternary complex the of of GDP from the complex in the presence of an excess of free is not as as that observed in the presence of GTP has a for the EF-1βγ subunits the exchange of GDP from G.M.C. Möller W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of to GTP for the stimulation of ValRS by EF-1α also that from be has not the of GTP to a in the ValRS·EF-1H complex that the of the ternary complex of nucleotide on the GDP exchange reaction catalyzed by EF-1H from the ValRS·EF-1H complex. The of GDP exchange from was at °C in the or in the presence of the EF-1H subunits from the ValRS·EF-1H complex. The reaction was by addition of GDP GTP or as described of to EF-1α addition of a nucleotide was as The that might for the observed EF-1α·GTP-dependent stimulation of ValRS activity that the is by of ValRS activity. We this possibility by to the aminoacylation mixture the of EF-1α, that is to in its with aminoacyl-tRNAs Biochem. Res. PubMed Scopus Google Scholar). Since EF-Tu has a for GTP for GDP K. M. J. Biochem. PubMed Scopus Google Scholar) and be with GTP by the nucleotide exchange subunits of EF-1H nucleotide exchange was by of the factor with an excess of GTP in the presence of and EF-1α·GTP was by EF-Tu·GTP in the aminoacylation no effect of EF-Tu·GTP on the ValRS aminoacylation activity was observed at of the factor μm) This suggested that the stimulation of synthesis catalyzed by the ValRS component of the ValRS·EF-1H complex is by a protein-protein interaction factors from for the of the native ValRS·EF-1H in the EF-1α·GTP stimulation of ValRS two ValRS that as free species were isolated from the complex. was previously that the of ValRS to with EF-1H is by elastase of the native enzyme of to a active truncated form of (20Bec G. Kerjan P. Waller J.P. J. Biol. Chem. 1994; 269: 2086-2092Abstract Full Text PDF PubMed Google Scholar). The acid extension of ValRS elastase is involved in the of the ValRS·EF-1H complex protein-protein interaction with the δ of EF-1H. native ValRS be from the EF-1H components in the presence of 0.5 m a (19Bec G. Waller J.P. J. Biol. Chem. 1989; 264: 21138-21143Abstract Full Text PDF PubMed Google Scholar). The ValRS species is isolated of its aminoacylation activity. two ValRS species were for their to be by EF-1α·GTP in the native ValRS·EF-1H species the truncated free ValRS, the native free ValRS, could be by the addition of EF-1α·GTP in the incubation mixture addition of the EF-1βγ subunits in the no effect on ValRS activity As the and subunits form a stable complex that the nucleotide from not to the isolated or ValRS species (20Bec G. Kerjan P. Waller J.P. J. Biol. Chem. 1994; 269: 2086-2092Abstract Full Text PDF PubMed Google Scholar). In the of the the ValRS·EF-1H complex be the the with the native ValRS form (20Bec G. Kerjan P. Waller J.P. J. Biol. Chem. 1994; 269: 2086-2092Abstract Full Text PDF PubMed Google in the of the and subunits, this nucleotide exchange factor not on EF-1α the to ValRS activity the association of ValRS with the β, γ, and δ subunits of EF-1H to be absolutely required to a functional interaction between the synthetase and elongation factor EF-1α. This that a of EF-1α·GTP and an essential for the of the synthetase activity. The presence in solution of a protein or with a high for the aminoacyl-tRNA the observed stimulation In the of ValRS, for of the aminoacyl-tRNA was to be the in the aminoacylation reaction D. J. PubMed Scopus Google addition of EF-1α, and GTP to have no effect not incubation of the multisynthetase complex from rabbit containing aminoacyl-tRNA synthetases (18Mirande M. Le Corre D. Waller J.P. Eur. J. Biochem. 1985; 147: 281-289Crossref PubMed Scopus (81) Google Scholar) in the presence of EF-1α·GTP not on or synthetases aminoacylation rates not The for tRNA in the aminoacylation reaction catalyzed by ValRS from the ValRS·EF-1H complex were in the presence or in the of EF-1α·GTP in the incubation were beef liver tRNA in pmol/A 260). The for tRNA of not by the addition of EF-1α·GTP in the aminoacylation the was found to be from to in the presence of we suggest that the effect of the interaction of ValRS·EF-1H with EF-1α·GTP is in the of ValRS, a that the of The stimulation of ValRS be in the presence of GTP not of This finding the of the ternary complex EF-1α·GTP·Val-tRNAVal for the stimulation mechanism. As is observed as as free EF-1α·GTP is in the incubation EF-1α is converted to ValRS is no that EF-1α not from GTP and the activity of EF-1α is by the aminoacyl-tRNA Parmeggiani A. Eur. J. Biochem. PubMed Scopus Google EF-1α not to be in this is possible to GTP by This GTP be as the nucleotide in its state complex. the activity of EF-1α is not involved in the stimulation of ValRS activity. ValRS is a acid protein Biochem. J. 1991; PubMed Scopus Google Scholar). As with its the enzyme a NH2-terminal polypeptide extension for its activity (20Bec G. Kerjan P. Waller J.P. J. Biol. Chem. 1994; 269: 2086-2092Abstract Full Text PDF PubMed Google Scholar). In have that this with the δ of EF-1H (20Bec G. Kerjan P. Waller J.P. J. Biol. Chem. 1994; 269: 2086-2092Abstract Full Text PDF PubMed Google Scholar). The finding that the native enzyme of the truncated ValRS species are to EF-1α stimulation also that the NH2-terminal extension of ValRS is not in a functional interaction with EF-1α. the NH2-terminal polypeptide extension of the synthetase, and the component of the multisynthetase complex with this S. M. FEBS Lett. 1996; PubMed Scopus (75) Google Scholar). This to the that these polypeptide could be involved in the of EF-1α to the complex. we were to effect of EF-1α·GTP on the activity of three components of the multisynthetase and we the possibility that a molecule could be involved to a functional interaction between EF-1α and a by the EF-1βγδ subunits in the of the ValRS·EF-1H complex. The of aminoacylation by ValRS requires the ValRS·EF-1H complex to EF-Tu for mammalian EF-1α, free ValRS the ValRS component of the ValRS·EF-1H complex. the isolated exchange factors, or the of ValRS by EF-1α. these results the direct connection between regulation of ValRS activity and the association of ValRS and EF-1α within a macromolecular assemblage of and The ValRS·EF-1H complex a structural for the functional interaction of ValRS with EF-1α. A is in ValRS is tightly associated to the β, γ, and δ subunits of EF-1H. EF-1α is to this is from the components on a column J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) or incubation with aminoacyl-tRNA and GTP (17Janssen G.M.C. van Damme H.T.F. Kriek J. Amons R. Möller W. J. Biol. Chem. 1994; 269: 31410-31417Abstract Full Text PDF PubMed Google of the ValRS·EF-1H complex exchange GTP for GDP from a molar excess of be EF-1α·GTP by the exchange factors to ValRS in the of synthetase at in In the ValRS component of the complex is to catalyze aminoacylation in the of GTP or of an excess of free EF-1α, that is the elongation factors are not functioning the functioning of ValRS and of the subunits is accompanied by a 2-fold increase in the Since is the EF-1α·GTP ValRS activity is the of a protein-protein interaction that a in ValRS, and the of from the that is the of these two reactions the direct transfer of the aminoacylated tRNA to the elongation with formation of the ternary complex EF-1α·GTP·Val-tRNAVal this coupling could be for the channeling of tRNA reported by Deutscher and (8Negrutskii B.S. Deutscher M.P. Proc. Natl Acad. Sci. U. S. A. 1991; 88: 4991-4995Crossref PubMed Scopus (157) Google Scholar, 10Stapulionis R. Deutscher M.P. Proc. Natl Acad. Sci. U. S. A. 1995; 92: 7158-7161Crossref PubMed Scopus (143) Google Scholar). a observed that tRNA or aminoacyl-tRNA are not for protein that is a channeled tRNA cycle in mammalian cells. In the of the ValRS results suggest that the of the aminoacylated tRNA from the synthetase could be controlled by the of free EF-1α, a for In that be that a of data that of the EF-1H and of the ValRS·EF-1H complexes are the of J. Biol. Chem. 1991; Full Text PDF PubMed Google G.M.C. Amons R. Möller W. J. Biol. Chem. Full Text PDF PubMed Google Scholar, E. 1994; PubMed Scopus Google Scholar, R. R. P. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of elongation factor is also observed in cells J. PubMed Google Scholar). The functional of these is could translational these have a on the stimulation of ValRS, and on tRNA channeling in vivo to be could be a to regulate the efficiency of tRNA delivery for protein synthesis. The from an are