Purification and properties of a high‐molecular‐mass complex between Val‐tRNA synthetase and the heavy form of elongation factor 1 from mammalian cells

Abstract

In extracts of various mammalian tissues obtained in the presence of protease inhibitors Val‐tRNA synthetase exists exclusively as a complex with a molecular mass of about 800 kDa. This complex was purified by gel filtration and two HPLC steps and contained five different polypeptides with molecular masses of 140, 50, 50, 40 and 30 kDa. The complex seems to have no tissue or species specificity, because preparations with identical polypeptide composition were obtained by the same method from rabbit liver and reticulocytes, and rat and beef liver. Four low‐molecular‐mass polypeptides were identified by two‐dimensional electrophoresis as subunits of the heavy form of elongation factor 1 (EF‐1H). The complex possesses the activity of EF‐1 in the poly(U)‐directed translation system, indicating that EF‐1H is an integral part of the complex. Gel filtration of the tissue extracts reveals three different peaks of EF‐1 activity, corresponding to EF‐1α, EF‐1H and the high‐molecular‐mass complex of Val‐tRNA synthetase and EF‐1H. All activity of Val‐tRNA synthetase and about 25% of EF‐1 activity are associated with the complex. Different forms of EF‐1 revealed no significant differences in the nucleotide‐binding properties, but the complex of Val‐tRNA synthetase with EF‐1H was 10 times more active in the poly(U)‐directed binding of Phe‐tRNA phe to ribosomes than EF‐1H. These result strongly suggest that the complex of Val‐tRNA synthetase with EF‐1H is a novel functionally active individual form of EF‐1.

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse, ancient protein super family using motif search and structural modeling

“…It has been shown that the interaction between EFly and EFlP involves the N-terminal portion of each of these proteins, which according to our findings, is related to GST (Van Damme et al, 1991). Human ValRS is present in the cell almost entirely as a complex with EFl, which accounts for about 25-50% of the total activity of this factor (Motorin et al, 1987(Motorin et al, , 1991Bec et al, 1989;Venemaet al, 1991). Recent experiments have shown that the N-terminal domain of ValRS, which may be an inactive homologue of the GST domain of EFly (see above), is responsible for the formation of the complex with EF1 (Beck et al.…”

The p18 component of the multisynthetase complex shares a protein motif with the β and γ subunits of eukaryotic elongation factor 1

“…The heavy form of EF-1 is a pentameric com- Table 1 Mixed oligonucleotide primers deduced from the amino acid sequence of the tryptic peptides of sheep pl8 Peptide and oligonucleotide sequences Name Sequence plex composed of the four subunits a, [3, 1, and 8 in a molar homology domain is also recovered in the NH2-terminal polyratio 2:1:1:1 [24]. EF-lc~ forms a ternary complex with amipeptide extension of human valyl-tRNA synthetase, an ennoacyl-tRNA and GTP, and is the donor of aminoacyl-tRNA zyme that forms a stable complex with EF-1H [3,4]. It was for ribosomal protein synthesis.…”

Occurrence of the aminoacyl‐tRNA synthetases in high‐molecular weight complexes correlates with the size of substrate amino acids

“…The activity of the factor is distributed between ''free'' EF-1A, heavy form of EF-1 (EF-1H) that contains eEF-1A associated with three other subunits (eEF1Ba, b, and c), and the Val-RS \ EF-1H complex. All these forms of EF-1 are functional in vitro [5], but their specificity toward aminoacyl-tRNAs has never been addressed.…”

“…The complex also contains three non-AARS proteins (p43, p38 and p18) with largely unclear function, although p43 is known to have nonspecific tRNA-binding activity [4]. A tenth AARS, valyl-tRNA synthetase (ValRS) does not participate in this complex, but forms a separate complex together with four subunits (eEF1A, eEF-1Ba, b, and c) of translation elongation factor 1 (EF-1) [5,6]. Multienzyme complexes typically unite polypeptides that are either involved in the same catalytic function or that catalyze consecutive reactions.…”