Bec G, Kerjan P, Waller J P
Laboratoire d'Enzymologie, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
J Biol Chem. 1994 Jan 21;269(3):2086-92.
Valyl-tRNA synthetase from mammalian cells is isolated exclusively as a complex with elongation factor (EF) 1H (the "heavy" form of eukaryotic EF-1, composed of subunits alpha, beta, gamma, and delta). In a previous study, the 140-kDa valyl-tRNA synthetase subunit dissociated from the purified rabbit liver complex was shown to display hydrophobic properties, unlike the corresponding yeast cytoplasmic enzyme of 125 kDa (Bec, G., and Waller, J.-P. (1989) J. Biol. Chem. 264, 21138-21143). Compared to the sequence of yeast cytoplasmic valyl-tRNA synthetase, that of the human enzyme displays an NH2-terminal extension of approximately 200 amino acid residues that bears strong sequence similarity to the NH2-terminal moiety of EF-1 gamma (Hsieh, S. L., and Campbell, R. D. (1991) Biochem. J. 278, 809-816). We now show that this NH2-terminal extension can be selectively excised by elastase treatment of the isolated rabbit valyl-tRNA synthetase, without impairing catalytic activity. To examine the role of the NH2-terminal extension of mammalian valyl-tRNA synthetase in complex formation and to identify the subunit(s) of EF-1H responsible for binding the enzyme, reconstitution experiments were undertaken. Native or truncated valyl-tRNA synthetases were incubated with the isolated EF-1 subunits beta gamma and delta, either separately or in combination, and the ensuing products were analyzed by chromatography on DEAE-Sepharose FF and Superose 6. The results demonstrate that the NH2-terminal extension of valyl-tRNA synthetase is required for complex formation and that the enzyme-binding site(s) resides on the EF-1 delta subunit. Moreover, although the EF-1 beta gamma binary complex does not bind valyl-tRNA synthetase, it is nevertheless required for assembly of a complex of defined quaternary structure by preventing the formation of high molecular weight aggregates generated in the presence of EF-1 delta alone.
哺乳动物细胞中的缬氨酰 - tRNA合成酶仅作为与延伸因子（EF）1H（真核生物EF - 1的“重”形式，由α、β、γ和δ亚基组成）的复合物被分离出来。在先前的一项研究中，从纯化的兔肝复合物中解离出来的140 kDa缬氨酰 - tRNA合成酶亚基显示出疏水特性，这与相应的125 kDa酵母细胞质酶不同（Bec, G., and Waller, J.-P. (1989) J. Biol. Chem. 264, 21138 - 21143）。与酵母细胞质缬氨酰 - tRNA合成酶的序列相比，人类酶的序列在NH2末端有一个约200个氨基酸残基的延伸，该延伸与EF - 1γ的NH2末端部分具有很强的序列相似性（Hsieh, S. L., and Campbell, R. D. (1991) Biochem. J. 278, 809 - 816）。我们现在表明，通过对分离的兔缬氨酰 - tRNA合成酶进行弹性蛋白酶处理，可以选择性地切除这个NH2末端延伸部分，而不会损害催化活性。为了研究哺乳动物缬氨酰 - tRNA合成酶NH2末端延伸在复合物形成中的作用，并确定负责结合该酶的EF - 1H亚基，我们进行了重组实验。将天然或截短的缬氨酰 - tRNA合成酶与分离的EF - 1亚基βγ和δ分别或组合孵育，然后通过DEAE - Sepharose FF和Superose 6柱层析分析所得产物。结果表明，缬氨酰 - tRNA合成酶的NH2末端延伸对于复合物形成是必需的，并且酶结合位点位于EF - 1δ亚基上。此外，尽管EF - 1βγ二元复合物不结合缬氨酰 - tRNA合成酶，但它对于形成具有确定四级结构的复合物是必需的，因为它可以防止仅在存在EF - 1δ时产生的高分子量聚集体的形成。
