Incorporation of the Arc1p tRNA-binding domain to the catalytic core of MetRS can functionally replace the yeast Arc1p-MetRS complex
Incorporation of the Arc1p tRNA-binding domain to the catalytic core of MetRS can functionally replace the yeast Arc1p-MetRS complex
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DOI：
10.1016/j.jmb.2008.06.044
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发表时间：
2008-09-05
影响因子：
5.6
通讯作者：
Simos, George
中科院分区：
文献类型：
--
作者：
Karanasios, Eleftherios;Boleti, Haralabia;Simos, George
甲硫酰-tRNA合成酶(MetRS)的催化核心在所有生命界中都是保守的，但根据物种来源的不同，往往与其N-端或C-端附加的非催化结构域相连。这些结构域通常有助于蛋白质-蛋白质或蛋白质-tRNA的相互作用，但对它们的确切生物学作用和进化目的知之甚少。酵母MetRs含有一个N端的附录，它介导了它与Arc1p的N端部分的相互作用。与Arc1p的联合作用控制MetRs的亚细胞分布。此外，Arc1p的C末端含有一个保守的tRNA结合域(TRBD)，这是依赖Arc1p刺激MetRs催化活性所必需的。同样的TRBD被发现直接与植物和线虫MetRs的催化域以及人的酪氨酰-tRNA合成酶融合。为了探讨在酵母中形成Arc1p-MetRS复合体的目的，我们测试了TRBD独立于Arc1p辅助MetRS功能的能力。我们通过构建嵌合蛋白MC-TRBD将TRBD直接连接到MetRS催化核心(MC)的C末端。分析了MC-TRBD的表达对酵母细胞生长、定位和体外氨酰化活性的影响，并与单独表达MC或野生型MetRs进行了比较。结果表明，MC-TRBD具有较高的酶活性，在体内可有效替代MetRS-Arc1p二元复合体。此外，MC-TRBD完全是细胞质的，在亚细胞定位方面也模拟了MetRS-Arc1p复合体。我们的结果表明，酵母MetRS的N末端附加域的唯一作用是介导与TRBD的间接结合，然而，当TRBD直接与催化的MetRS核心融合时，它也可以在体内有效地发挥作用。(C)2008爱思唯尔有限公司。保留所有权利。
The catalytic core of methionyl-tRNA synthetase (MetRS) is conserved among all life kingdoms but, depending on species origin, is often linked to noncatalytic domains appended to its N- or C-terminus. These domains usually contribute to protein-protein or protein-tRNA interactions but their exact biological role and evolutionary purpose is poorly understood. Yeast MetRS contains an N-terminal appendix that mediates its interaction with the N-terminal part of Arc1p. Association with Arc1p controls the subcellular distribution of MetRS. Furthermore, the C-terminal part of Arc1p harbors a conserved tRNA-binding domain (TRBD) required for the Arc1p-dependent stimulation of the catalytic activity of MetRS. The same TRBD is found directly fused to catalytic domains of plant and nematode MetRS as well as human tyrosyl-tRNA synthetase. To investigate the purpose of Arc1p-MetRS complex formation in yeast, we tested the ability of TRBD to assist the function of MetRS independently of Arc1p. We attached the TRBD directly to the C-terminus of the MetRS catalytic core (MC) by constructing the chimeric protein MC-TRBD. The effect of MC-TRBD expression on yeast cell growth as well as its localization and in vitro aminoacylation activity were analyzed and compared to that of MC alone or wild-type MetRS, both in the absence or presence of Arc1p. We show that MC-TRBD exhibits improved enzymatic activity and can effectively substitute the MetRS-Arc1p binary complex in vivo. Moreover, MC-TRBD, being exclusively cytoplasmic, also mimics the MetRS-Arc1p complex in terms of subcellular localization. Our results suggest that the sole role of the N-terminal appended domain of yeast MetRS is to mediate the indirect association with the TRBD, which, nevertheless, can also function effectively in vivo when directly fused to the catalytic MetRS core. (C) 2008 Elsevier Ltd. All rights reserved.