Characterization of p43(ARF), a derivative of the p43 component of multiaminoacyl-tRNA synthetase complex released during apoptosis.

In human, nine aminoacyl tRNA synthetases are associated with the three auxiliary proteins, p18, p38, and p43, to form a stable multiprotein complex. The p43 component, which has a potent tRNA binding capacity, is associated to the complex via its N-terminal moiety. This protein is also the precursor of the endothelial monocyte-activating polypeptide II (p43(EMAPII), corresponding to the C-terminal moiety of p43), a cytokine generated during apoptosis. Here we examined the cellular pathway that, starting from the p43 subunit of the complex, leads to this extracellular cytokine. We identified a new intermediate in this pathway, named p43(ARF) for Apoptosis-released Factor. This intermediate is produced in cellulo by proteolytic cleavage of endogenous p43 and is rapidly recovered in the culture medium. This p43 derivative was purified from the medium of human U937 cells subjected to serum starvation. It contains 40 additional N-terminal amino acid residues as compared with the cytokine p43(EMAPII) and may be generated by a member of the matrix metalloproteinase family. Recombinant p43(ARF) is a monomer in solution and binds tRNA with a Kd of approximately 6 nM, 30-fold lower than that of p43. Highly purified p43(ARF) or p43(EMAPII) do not stimulate the expression of E-selectin by human umbilical vein endothelial cells. Our results suggest that the cleavage of p43 and its cellular delocalization, and thus the release of this tRNA binding subunit from the complex, is one of the molecular mechanisms leading to the shut down of protein synthesis in apoptosis.

Determinants in tRNA for activation of arginyl-tRNA synthetase: evidence that tRNA flexibility is required for the induced-fit mechanism.

Arginyl-tRNA synthetase (ArgRS) catalyzes formation of arginyl-adenylate in a tRNA-dependent reaction. Previous studies have revealed that conformational changes occur upon tRNA binding. In this study, we analyzed the sequence and structural features of tRNA that are essential to activate the catalytic center of mammalian arginyl-tRNA synthetase. Here, tRNA variants with different activator potential are presented. The three regions that are crucial for activation of ArgRS are the terminal adenosine, the D-loop, and the anticodon stem-loop of tRNA. The Add-1 N-terminal domain of ArgRS, which has the very unique property among aminoacyl-tRNA synthetases to interact with the D-loop in the corner of the convex side of tRNA, has an essential role in anchoring tRNA and participating in tRNA-induced amino acid activation. The results suggest that locking the acceptor extremity, the anticodon loop, and the D-loop of tRNA on the catalytic, anticodon-binding, and Add-1 domains of ArgRS also requires some flexibility of the tRNA molecule, provided by G:U base pairs, to achieve the productive conformation of the active site of the enzyme by induced fit.

The tRNA-interacting factor p43 associates with mammalian arginyl-tRNA synthetase but does not modify its tRNA aminoacylation properties.

Arginyl-tRNA synthetase (ArgRS) is one of the nine synthetase components of a multienzyme complex containing three auxiliary proteins as well. We previously established that the N-terminal moiety of the auxiliary protein p43 associates with the N-terminal, eukaryotic-specific polypeptide extension of ArgRS. Because p43 is homologous to Arc1p, a yeast general RNA-binding protein that associates with MetRS and GluRS and plays the role of tRNA-binding cofactor in the aminoacylation reaction, we analyzed the functional significance of p43-ArgRS association. We had previously showed that full-length ArgRS, corresponding to the ArgRS species associated within the multisynthetase complex, and ArgRS with a deletion of 73 N-terminal amino acid residues, corresponding to a free species of ArgRS, both produced in yeast, have similar catalytic parameters (Lazard, M., Kerjan, P., Agou, F., and Mirande, M. (2000) J. Mol. Biol. 302, 991-1004). However, a recent study had suggested that association of p43 to ArgRS reduces the apparent K(M) of ArgRS to tRNA (Park, S. G., Jung, K. H., Lee, J. S., Jo, Y. J., Motegi, H., Kim, S., and Shiba, K. (1999) J. Biol. Chem. 274, 16673-16676). In this study, we analyzed in detail, by gel retardation assays and enzyme kinetics, the putative role of p43 as a tRNA-binding cofactor of ArgRS. The association of p43 with ArgRS neither strengthened tRNA-binding nor changed kinetic parameters in the amino acid activation or in the tRNA aminoacylation reaction. Furthermore, selective removal of the C-terminal RNA-binding domain of p43 from the multisynthetase complex did not affect kinetic parameters for ArgRS. Therefore, p43 has a dual function. It promotes association of ArgRS to the complex via its N-terminal domain, but its C-terminal RNA-binding domain may act as a tRNA-interacting factor for an as yet unidentified component of the complex.