Kobayashi Nobuaki, Yang Jun, Ueda Atsuhisa, Suzuki Takeyuki, Tomaru Kouji, Takeno Mitsuhiro, Okuda Kenji, Ishigatsubo Yoshiaki
Department of Internal Medicine and Clinical Immunology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama-City 236-0004, Japan.
Gene. 2007 Jul 15;396(2):236-47. doi: 10.1016/j.gene.2007.02.032. Epub 2007 Mar 24.
Ran-binding protein in microtubule organising centre (RanBPM) was originally isolated as a protein that binds to the small GTPase Ran. Recently our group and other groups reported that RanBPM was associated with several proteins and composed a large protein complex. Here, we used tandem MS with an antibody against RanBPM to purify this complex from a soluble extract of HEK293 cells: we identified Muskelin, p48EMLP, p44CTLH, and the novel armadillo-repeat proteins ARMC8alpha and ARMC8beta as components. In RanBPM, Muskelin, p48EMLP, and p44CTLH we found LisH/CTLH motifs, which are present in proteins involved in microtubule dynamics, cell migration, nucleokinesis, and chromosome segregation. We renamed the 20S large protein complex the CTLH complex. The N-terminal 364 amino acids of ARMC8alpha and ARMC8beta were completely conserved, suggesting that these proteins are probably alternatively spliced products from the same gene. We confirmed the in vivo association of each component by co-immunoprecipitation assays with Cos-7 cells in which these components were exogenously overexpressed. A pull-down assay with bacterially-expressed Twa1 revealed binding of each in vitro-translated component to Twa1. Finally, we confirmed the cellular localization of these proteins. Taken together, our results reveal that RanBPM, ARMC8alpha, ARMC8beta, Muskelin, p48EMLP, and p44CTLH form complexes in cells.
微管组织中心的Ran结合蛋白（RanBPM）最初是作为一种与小GTP酶Ran结合的蛋白质被分离出来的。最近，我们小组和其他小组报道RanBPM与几种蛋白质相关联，并组成了一个大型蛋白质复合物。在这里，我们使用串联质谱结合抗RanBPM抗体从HEK293细胞的可溶性提取物中纯化该复合物：我们鉴定出肌动蛋白结合蛋白（Muskelin）、p48EMLP、p44CTLH以及新型犰狳重复蛋白ARMC8α和ARMC8β为其组成成分。在RanBPM、Muskelin、p48EMLP和p44CTLH中，我们发现了LisH/CTLH基序，这些基序存在于参与微管动力学、细胞迁移、核运动和染色体分离的蛋白质中。我们将这个20S大型蛋白质复合物重新命名为CTLH复合物。ARMC8α和ARMC8β的N端364个氨基酸完全保守，这表明这些蛋白质可能是同一基因的可变剪接产物。我们通过在Cos-7细胞中外源过表达这些成分的共免疫沉淀实验，证实了各成分在体内的关联性。用细菌表达的Twa1进行的下拉实验揭示了每个体外翻译成分与Twa1的结合。最后，我们证实了这些蛋白质在细胞内的定位。综上所述，我们的结果表明RanBPM、ARMC8α、ARMC8β、Muskelin、p48EMLP和p44CTLH在细胞中形成复合物。
