Macromolecular assemblage of aminoacyl-tRNA synthetases: Identification of protein-protein interactions and characterization of a core protein
Macromolecular assemblage of aminoacyl-tRNA synthetases: Identification of protein-protein interactions and characterization of a core protein
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DOI：
10.1006/jmbi.1998.2316
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发表时间：
1999-01-08
影响因子：
5.6
通讯作者：
Mirande, M
中科院分区：
文献类型：
--
作者：
Quevillon, S;Robinson, JC;Mirande, M
在真核生物中，从苍蝇到人类，九种氨基酰基trna合成酶构成了一个具有明确和保守结构组织的多酶复合体。这种普遍存在的多蛋白组合包括独特的双功能氨基酰基- trna合成酶，谷氨酰基-脯氨酸- trna合成酶，以及单特异性异亮基，亮基，谷氨酰基，蛋氨酸基，赖氨酸基，精氨酸基和天冬氨酸- trna合成酶。三种表观分子质量分别为18、38和43 kDa的辅助蛋白总是与该复合物的九种tRNA合成酶组分相关联。作为研究这个大分子组装的分子和功能组织的一部分，我们分离了编码p38非合成酶成分的cDNA并确定了其功能。根据对完整基因组序列的检查，320个氨基酸残基编码的蛋白质在酵母、细菌和古细菌中没有同源物。p38蛋白是一种中度疏水蛋白，显示一个假定的亮氨酸-拉链基序，并与参与蛋白-蛋白相互作用的蛋白结构域共享序列模式。我们使用酵母双杂交系统来记录复合物组分之间的蛋白质连接。我们对相互作用蛋白进行了详尽的搜索，涉及该复合物的11个组分中的10个。21个蛋白质对已被明确识别，导致多合成酶复合物亚基的拓扑排列的全局视图。特别是，p38被发现与自身结合形成二聚体，但也与p43结合，与I类tRNA合成酶ArgRS和GLnRS，与II类合成酶AspRS和LysRS，以及与双功能的GluProRS结合。我们生成了一系列缺失突变体来定位p38介导鉴定的相互作用的区域。绘制p38的相互作用结构域显示了p38与其不同蛋白伴侣的特异性关联。这些发现表明，p38在真核tRNA合成酶复合物亚基的组装中起着关键作用，而迄今为止在缺乏多合成酶复合物的生物体中尚未发现其同源蛋白。(C) 1999学术出版社。
In eukaryotes, from fly to human, nine aminoacyl-tRNA synthetases contribute a multienzyme complex of defined and conserved structural organization. This ubiquitous multiprotein assemblage comprises a unique bifunctional aminoacyl-tRNA synthetase, glutamyl-prolyl-tRNA synthetase, as well as the monospecific isoleucyl, leucyl, glutaminyl, methionyl, lysyl, arginyl, and aspartyl-tRNA synthetases. Three auxiliary proteins of apparent molecular masses of 18, 38 and 43 kDa are invariably associated with the nine tRNA synthetase components of the complex. As part of an inquiry into the molecular and functional organization of this macromolecular assembly, we isolated the cDNA encoding the p38 non-synthetase component and determined its function. The 320 amino acid residue encoded protein has been shown to have no homolog in yeast, bacteria and archaea, according to the examination of the complete genomic sequences available. The p38 protein is a moderately hydrophobic protein, displays a putative leucine-zipper motif, and shares a sequence pattern with protein domains that are involved in protein-protein interactions. We used the yeast two-hybrid system to register protein connections between components of the complex. We performed an exhaustive search of interactive proteins, involving 10 of the 11 components of the complex. Twenty-one protein pairs have been unambiguously identified, leading to a global view of the topological arrangement of the subunits of the multisynthetase complex. In particular, p38 was found to associate with itself to form a dimer, but also with p43, with the class I tRNA synthetases ArgRS and GLnRS, with the class II synthetases AspRS and LysRS, and with the bifunctional GluProRS. We generated a series of deletion mutants to localize the regions of p38 mediating the identified interactions. Mapping the interactive domains in p38 showed the specific association of p38 with its different protein partners. These findings suggest that p38, for which no homologous protein has been identified to date in organisms devoid of multisynthetase complexes, plays a pivotal role for the assembly of the subunits of the eukaryotic tRNA synthetase complex. (C) 1999 Academic Press.