CHMP4b is a major binding partner of the ALG-2-interacting protein Alix among the three CHMP4 isoforms
CHMP4b is a major binding partner of the ALG-2-interacting protein Alix among the three CHMP4 isoforms
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DOI：
10.1016/j.abb.2003.09.038
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发表时间：
2004-01-01
影响因子：
3.9
通讯作者：
Maki, M
中科院分区：
文献类型：
--
作者：
Katoh, K;Shibata, H;Maki, M
ALG-2相互作用蛋白Alix最近被证明与CHMP4b有关，CHMP4b是酵母Snf7p(也称为Vps32p)的人类同源物，也是小卷曲蛋白CHMP家族的成员，CHMP参与多囊泡体的分选。除了先前分离的两种CHMP4蛋白(CHMP4a和CHMP4b)的cDNA外，我们还克隆了CHMP4亚家族的一个新成员(命名为CHMP4c)的cDNA。Northern印迹分析显示，这三种CHMP4亚型的mRNAs在人体组织中的表达模式不同。与CHMP4a和CHMP4c转录本相比，CHMP4b在所有被测试的12个组织中的表达水平都更高，特别是在心肌和骨骼肌中。用HEK293细胞表达的CHMP4蛋白的裂解产物和Alix与绿色荧光蛋白的融合，通过共沉淀实验检测每种CHMP4亚型与Alix的相互作用。此外，利用截短Alix(氨基酸1-423)的重组谷胱甘肽S转移酶(GST)与硫氧还蛋白标记的CHMP4蛋白的融合蛋白，通过GST下拉实验检测到CHMP4b与CHMP4亚型的直接相互作用，其中CHMP4b显示出比其他CHMP4亚型更强的相互作用。这些结果表明，在三种CHMP4亚型中，CHMP4b是Alix的主要结合伙伴。(C)2003 Elsevier Inc.保留所有权利。
The ALG-2-interacting protein Alix has recently been demonstrated to associate with CHMP4b that is a human homologue of yeast Snf7p (also named Vps32p) and a member of the family of small coiled-coil proteins named CHMP implicated in playing roles in multivesicular body sorting. In addition to the previously isolated cDNAs for two CHMP4 proteins (CHMP4a and CHMP4b), we isolated a cDNA for a new member of the CHMP4 subfamily (designated CHMP4c). Northern blot analyses revealed different expression patterns of the mRNAs for the three CHMP4 isoforms in human tissues. CHMP4b messages were expressed at higher levels in all 12 tissues tested in comparison with the CHMP4a and CHMP4c transcripts, particularly in heart and skeletal muscle. The interaction with Alix was detected for each CHMP4 isoform by co-inummoprecipitation experiments using lysates of HEK293 cells expressing each epitope-tagged CHMP4 protein and Alix fused with green fluorescent protein. Further, using recombinant glutathione S-transferase (GST) fusion protein of truncated Alix (amino acids 1-423) and thioredoxin-tagged CHMP4 proteins, the direct interactions were detected by a GST pull-down assay, where CHMP4b showed a stronger interaction than other CHMP4 isoforms. These results suggest that CHMP4b is a major binding partner of Alix among the three CHMP4 isoforms. (C) 2003 Elsevier Inc. All rights reserved.