Biomedical Research Institute, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, Korea ; School of Life Sciences and Biotechnology, Korea University, Seongbuk-gu, Seoul, Korea.
PLoS One. 2013 Dec 2;8(12):e81734. doi: 10.1371/journal.pone.0081734. eCollection 2013.
Twenty different aminoacyl-tRNA synthetases (ARSs) link each amino acid to their cognate tRNAs. Individual ARSs are also associated with various non-canonical activities involved in neuronal diseases, cancer and autoimmune diseases. Among them, eight ARSs (D, EP, I, K, L, M, Q and RARS), together with three ARS-interacting multifunctional proteins (AIMPs), are currently known to assemble the multi-synthetase complex (MSC). However, the cellular function and global topology of MSC remain unclear. In order to understand the complex interaction within MSC, we conducted affinity purification-mass spectrometry (AP-MS) using each of AIMP1, AIMP2 and KARS as a bait protein. Mass spectrometric data were funneled into SAINT software to distinguish true interactions from background contaminants. A total of 40, 134, 101 proteins in each bait scored over 0.9 of SAINT probability in HEK 293T cells. Complex-forming ARSs, such as DARS, EPRS, IARS, Kars, LARS, MARS, QARS and RARS, were constantly found to interact with each bait. Variants such as, AIMP2-DX2 and AIMP1 isoform 2 were found with specific peptides in KARS precipitates. Relative enrichment analysis of the mass spectrometric data demonstrated that TARSL2 (threonyl-tRNA synthetase like-2) was highly enriched with the ARS-core complex. The interaction was further confirmed by coimmunoprecipitation of TARSL2 with other ARS core-complex components. We suggest TARSL2 as a new component of ARS core-complex.
二十种不同的氨酰-tRNA 合成酶 (ARSs) 将每种氨基酸与其相应的 tRNA 连接起来。个别 ARSs 还与涉及神经疾病、癌症和自身免疫性疾病的各种非典型活性有关。其中，八种 ARSs（D、EP、I、K、L、M、Q 和 RARS）以及三种 ARS 相互作用的多功能蛋白（AIMPs），目前已知可组装多合成酶复合物（MSC）。然而，MSC 的细胞功能和全局拓扑结构仍不清楚。为了了解 MSC 内的复杂相互作用，我们使用 AIMP1、AIMP2 和 KARS 中的每一种作为诱饵蛋白进行了亲和纯化-质谱（AP-MS）。质谱数据被导入到 SAINT 软件中，以区分真实的相互作用和背景污染物。在 HEK 293T 细胞中，每个诱饵蛋白的 40、134、101 种蛋白质的 SAINT 概率评分均超过 0.9。不断发现与每种诱饵蛋白相互作用的形成复合物的 ARSs，如 DARS、EPRS、IARS、Kars、LARS、MARS、QARS 和 RARS。还发现了 AIMP2-DX2 和 AIMP1 同工型 2 等变体，它们在 KARS 沉淀中具有特定的肽段。质谱数据的相对富集分析表明，TARSL2（苏氨酰-tRNA 合成酶样-2）与 ARS 核心复合物高度富集。通过与其他 ARS 核心复合物成分的共免疫沉淀进一步证实了这种相互作用。我们建议 TARSL2 作为 ARS 核心复合物的一个新组成部分。
