Penta-EF-hand protein ALG-2 functions as a Ca2+-dependent adaptor that bridges Alix and TSG101
Penta-EF-hand protein ALG-2 functions as a Ca2+-dependent adaptor that bridges Alix and TSG101
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DOI：
10.1016/j.bbrc.2009.06.015
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发表时间：
2009-08-14
影响因子：
3.1
通讯作者：
Maki, Masatoshi
中科院分区：
文献类型：
--
作者：
Okumura, Mayumi;Ichioka, Fumitaka;Maki, Masatoshi
已知阿利克斯和TSG 101相互作用，它们具有富含Pro的区域，这些区域依赖于ALG-2 Ca 2+结合。我们使用Strep标记的阿利克斯及其各种突变体通过下拉测定研究了ALG-2在Alix-TSG 101缔合中的作用。ALG-2结合位点是使用HEK 293 T细胞的TSG 101的Ca 2+依赖性下拉所需的，而PSAP序列（TSG 101的UEV结构域的结合基序）是不稳定的。使用ALG-2敲低细胞未观察到Alix-TSG 101缔合，但通过在测定混合物中加入纯化的重组ALG-2蛋白而变得可检测。mGFP融合ALG-2的外源表达也恢复了Strep-Alix的下拉能力，但选择性剪接的较短ALG-2同种型和二聚化缺陷突变体是不称职的。基于X-射线晶体结构模型显示ALG-2二聚体的每个分子中存在一个配体结合位点，我们提出负载Ca 2+的ALG-2桥阿利克斯和TSC 101作为接头蛋白。(C)2009爱思唯尔公司All rights reserved.
Alix and TSG101, known to physically interact with each other, have Pro-rich regions that are bound by ALG-2 Ca2+-dependently. We investigated the role of ALG-2 in the Alix-TSG101 association by pulldown assays using Strep-tagged Alix and its various mutants. The ALG-2-binding site was required for the Ca2+-dependent pulldown of TSG101 using HEK293T cells, whereas the PSAP sequence, a binding motif for the UEV domain of TSG101, was dispensable. Alix-TSG101 association was not observed using ALG-2-knock-down cells but became detectable by addition of the purified recombinant ALG-2 protein in the assay mixtures. Exogenous expression of mGFP-fused ALG-2 also restored the pulldown capability of Strep-Alix, but an alternatively spliced shorter ALG-2 isoform and a dimerization-defective mutant were incompetent. Based on the X-ray crystal structure model showing the presence of one ligand-binding site in each molecule of an ALG-2 dimer, we propose that Ca2+-loaded ALG-2 bridges Alix and TSC101 as an adaptor protein. (C) 2009 Elsevier Inc. All rights reserved.