Boris Negrutskii

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

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.

Dissection of the Structural Organization of the Aminoacyl-tRNA Synthetase Complex

The spatio-temporal organization of proteins within the cytoplasm of eukaryotic cells rests in part on the assembly of stable and transient multiprotein complexes. Here we examined the assembly of the multiaminoacyl-tRNA synthetase complex (MARS) in human cells. This complex contains nine aminoacyl-tRNA synthetases and three auxiliary proteins and is a hallmark of metazoan species. Isolation of the complexes has been performed by tandem affinity purification from human cells in culture. To understand the rules of assembly of this particle, expression of the three nonsynthetase components of MARS, p18, p38, and p43, was blocked by stable small interfering RNA silencing. The lack of these components was not lethal for the cells, but cell growth was slightly reduced. The residual complexes that could form in vivo in the absence of the auxiliary proteins were isolated by tandem affinity purification. From the repertoire of the subcomplexes that could be isolated, a comprehensive map of protein-protein interactions mediating complex assembly is deduced. The data are consistent with a structural role of the three nonsynthetase components of MARS, with p38 connecting two subcomplexes that may form in the absence of p38.

Dynamic Organization of Aminoacyl-tRNA Synthetase Complexes in the Cytoplasm of Human Cells

The localization in space and in time of proteins within the cytoplasm of eukaryotic cells is a central question of the cellular compartmentalization of metabolic pathways. The assembly of proteins within stable or transient complexes plays an essential role in this process. Here, we examined the subcellular localization of the multi-aminoacyl-tRNA synthetase complex in human cells. The sequestration of its components within the cytoplasm rests on the presence of the eukaryotic-specific polypeptide extensions that characterize the human enzymes, as compared with their prokaryotic counterparts. The cellular mobility of several synthetases, assessed by measuring fluorescence recovery after photobleaching, suggested that they are not freely diffusible within the cytoplasm. Several of these enzymes, isolated by tandem affinity purification, were copurified with ribosomal proteins and actin. The capacity of aminoacyl-tRNA synthetases to interact with polyribosomes and with the actin cytoskeleton impacts their subcellular localization and mobility. Our observations have conceptual implications for understanding how translation machinery is organized in vivo.

Rabbit translation elongation factor 1α stimulates the activity of homologous aminoacyl-tRNA synthetase

Functional and structural sequestration of aminoacyl‐tRNA has been recently found in eukaryotic cells and the aminoacyl‐tRNA channeling has been suggested [B.S. Negrutskii et al., Proc. Natl. Acad. Sci. 91 (1994) 964–968], but molecular details and mechanism of the process remained unclear. In this paper we have verified a possible interaction between rabbit aminoacyl‐tRNA synthetase and homologous translation elongation factor 1α (EF‐1α), the proteins which may play a role of sequential components involved into the transfer of the aminoacyl‐tRNA along the protein synthetic metabolic chain. The stimulation of the phenylalanyl‐tRNA synthetase activity by EF‐1α is found. The effect is shown to be specific towards the origin of tRNA and elongation factor molecules. The data obtained favor the direct transfer mechanism of the aminoacyl‐tRNA channeling process during eukaryotic protein synthesis.

Evidence for the formation of an unusual ternary complex of rabbit liver EF‐1α with GDP and deacylated tRNA

Eukaryotic translation elongation factor 1α is known to interact in GTP‐bound form with aminoacyl‐tRNA promoting its binding to the ribosome. In this paper another ternary complex [EF‐1α*GDP*deacylated tRNA], never considered in widely accepted elongation schemes, is reported for the first time. The formation of this unusual complex, postulated earlier (FEBS Lett. (1996) 382, 18–20), has been detected by four independent methods. [EF‐1α*GDP]‐interacting sites are located in the acceptor stem, TψC stem and TψC loop of tRNAPhe and tRNALeu molecules. Both tRNA and EF‐1α are found to undergo certain conformational changes during their interaction. The ability of EF‐1α to form a complex with deacylated tRNA indicates that the factor may perform an important role in tRNA and aminoacyl‐tRNA channeling in higher eukaryotic cells.

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon–anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the ‘GTP’- and ‘GDP’-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.

Caenorhabditis elegans Evolves a New Architecture for the Multi-aminoacyl-tRNA Synthetase Complex

MARS is an evolutionary conserved supramolecular assembly of aminoacyl-tRNA synthetases found in eukaryotes. This complex was thought to be ubiquitous in the deuterostome and protostome clades of bilaterians because similar complexes were isolated from arthropods and vertebrates. However, several features of the component enzymes suggested that in the nematode Caenorhabditis elegans, a species grouped with arthropods in modern phylogeny, this complex might not exist, or should display a significantly different structural organization. C. elegans was also taken as a model system to study in a multicellular organism amenable to experimental approaches, the reason for existence of these supramolecular entities. Here, using a proteomic approach, we have characterized the components of MARS in C. elegans. We show that this organism evolved a specific structural organization of this complex, which contains several bona fide components of the MARS complexes known so far, but also displays significant variations. These data highlight molecular evolution events that took place after radiation of bilaterians. Remarkably, it shows that expansion of MARS assembly in metazoans is not linear, but is the result of additions but also of subtractions along evolution. We then undertook an experimental approach, using inactivation of the endogenous copy of methionyl-tRNA synthetase by RNAi and expression of transgenic variants, to understand the role in complex assembly and the in vivo functionality, of the eukaryotic-specific domains appended to aminoacyl-tRNA synthetases. We show that rescue of the worms and assembly of transgenic variants into MARS rest on the presence of these appended domains.

From global phosphoproteomics to individual proteins: the case of translation elongation factor eEF1A.

Phosphoproteomics is often aimed at deciphering the modified components of signaling pathways in certain organisms, tissues and pathologies. Phosphorylation of housekeeping proteins, albeit important, usually attracts less attention. Here, we provide targeted analysis of eukaryotic translation elongation factor 1A (eEF1A), which is the main element of peptide elongation machinery. There are 97% homologous A1 and A2 isoforms of eEF1A; their expression in mammalian tissues is mutually exclusive and differentially regulated in development. The A2 isoform reveals proto-oncogenic properties and specifically interacts with some cellular proteins. Several tyrosine residues shown experimentally to be phosphorylated in eEF1A1 are hardly solution accessible, so their phosphorylation could be linked with structural rearrangement of the protein molecule. The possible role of tyrosine phosphorylation in providing the background for structural differences between the ‘extended’ A1 isoform and the compact oncogenic A2 isoform is discussed. The ‘road map’ for targeted analysis of any protein of interest using phosphoproteomics data is presented.

Unbalanced expression of the translation complex eEF1 subunits in human cardioesophageal carcinoma

Eur J Clin Invest 2011; 41 (3): 269–276

Methionyl‐tRNA synthetase from Caenorhabditis elegans: A specific multidomain organization for convergent functional evolution

Methionyl‐tRNA synthetase (MetRS) is a multidomain protein that specifically binds tRNAMet and catalyzes the synthesis of methionyl‐tRNAMet. The minimal, core enzyme found in Aquifex aeolicus is made of a catalytic domain, which catalyzes the aminoacylation reaction, and an anticodon‐binding domain, which promotes tRNA–protein association. In eukaryotes, additional domains are appended in cis or in trans to the core enzyme and increase the stability of the tRNA–protein complexes. Eventually, as observed for MetRS from Homo sapiens, the C‐terminal appended domain causes a slow release of aminoacyl‐tRNA and establishes a limiting step in the global aminoacylation reaction. Here, we report that MetRS from the nematode Caenorhabditis elegans displays a new type of structural organization. Its very C‐terminal appended domain is related to the oligonucleotide binding‐fold‐based tRNA‐binding domain (tRBD) recovered at the C‐terminus of MetRS from plant, but, in the nematode enzyme, this domain is separated from the core enzyme by an insertion domain. Gel retardation and tRNA aminoacylation experiments show that MetRS from nematode is functionally related to human MetRS despite the fact that their appended tRBDs have distinct structural folds, and are not orthologs. Thus, functional convergence of human and nematode MetRS is the result of parallel and convergent evolution that might have been triggered by the selective pressure to invent processivity of tRNA handling in translation in higher eukaryotes.