Mirande M, Lazard M, Martinez R, Latreille M T
Laboratoire d'Enzymologie du Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Eur J Biochem. 1992 Feb 1;203(3):459-66. doi: 10.1111/j.1432-1033.1992.tb16570.x.
Aspartyl-tRNA synthetase from higher eukaryotes is a component of a multienzyme complex comprising nine aminoacyl-tRNA synthetases. The cDNA encoding cytoplasmic rat liver aspartyl-tRNA synthetase was previously cloned and sequenced. This work reports the identification of structural features responsible for its association within the multisynthetase complex. Mutant and chimeric proteins have been expressed in mammalian cells and their structural behavior analyzed. A wild-type rat liver aspartyl-tRNA synthetase, expressed in Chinese hamster ovary (CHO) cells, associates within the complex from CHO cells, whereas a mutant enzyme with a deletion of 34 amino acids from its amino-terminal extremity does not. A chimeric enzyme, made of the amino-terminal moiety of rat liver aspartyl-tRNA synthetase fused to the catalytic domain of yeast lysyl-tRNA synthetase, has been expressed in Lys-101 cells, a CHO cell line with a temperature-sensitive lysyl-tRNA synthetase. The fusion protein is stable in vivo, does not associate within the multisynthetase complex and cannot restore normal growth of the mutant cells. These results establish that the 3.7-kDa amino-terminal moiety of mammalian aspartyl-tRNA synthetase mediates its association with the other components of the complex. In addition, the finding that yeast lysyl-tRNA synthetase cannot replace the aspartyl-tRNA synthetase component of the mammalian complex, indicates that interactions between neighbouring enzymes also play a prominent role in stabilization of this multienzyme structure and strengthened the view that the multisynthetase complex is a discrete entity with a well-defined structural organization.
高等真核生物的天冬氨酰 - tRNA合成酶是一种多酶复合物的组成成分，该复合物包含九种氨酰 - tRNA合成酶。先前已克隆并测序了编码大鼠肝脏细胞质天冬氨酰 - tRNA合成酶的cDNA。这项工作报道了负责其在多合成酶复合物中缔合的结构特征的鉴定。突变体和嵌合蛋白已在哺乳动物细胞中表达，并对其结构行为进行了分析。在中国仓鼠卵巢（CHO）细胞中表达的野生型大鼠肝脏天冬氨酰 - tRNA合成酶可在CHO细胞的复合物中缔合，而从其氨基末端缺失34个氨基酸的突变酶则不能。由大鼠肝脏天冬氨酰 - tRNA合成酶的氨基末端部分与酵母赖氨酰 - tRNA合成酶的催化结构域融合而成的嵌合酶已在Lys - 101细胞中表达，Lys - 101细胞系是一种具有温度敏感性赖氨酰 - tRNA合成酶的CHO细胞系。该融合蛋白在体内稳定，不在多合成酶复合物中缔合，也不能恢复突变细胞的正常生长。这些结果表明，哺乳动物天冬氨酰 - tRNA合成酶的3.7 kDa氨基末端部分介导了其与复合物其他组分的缔合。此外，酵母赖氨酰 - tRNA合成酶不能替代哺乳动物复合物中天冬氨酰 - tRNA合成酶组分这一发现表明，相邻酶之间的相互作用在这种多酶结构的稳定中也起着重要作用，并强化了多合成酶复合物是具有明确结构组织的离散实体这一观点。
