Identification of the T-complex protein as a binding partner for newly synthesized cytoplasmic dynein intermediate chain 2
Identification of the T-complex protein as a binding partner for newly synthesized cytoplasmic dynein intermediate chain 2
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DOI：
10.1016/j.bbrc.2015.11.082
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发表时间：
2016-01-01
影响因子：
3.1
通讯作者：
Catling, Andrew D.
中科院分区：
文献类型：
--
作者：
Ozdemir, Aysun;Machida, Kodai;Catling, Andrew D.
细胞质动力蛋白是真核细胞中一种具有多种功能的大分子马达复合物。动力蛋白在细胞器和有丝分裂的细胞内运输中起重要作用，部分通过动力蛋白中间链2（IC-2）亚基和结合特定货物的衔接蛋白之间的相互作用介导。在识别IC-2的磷酸化依赖性结合伴侣的实验中，我们反而识别了磷酸化独立的结合伴侣，即含有T复合物蛋白1（CCT）的胞质伴侣。CCT由八个亚基（CCT 1 -8）组成，并促进新合成蛋白质子集的折叠。我们在免疫共沉淀实验中证实了IC-2与CCT 5和CCT 8之间的相互作用，并确定IC-2的C-末端半部分对于结合CCT 8是必要且足够的。有趣的是，IC-2和CCT的共免疫沉淀被先前的放线菌酮处理细胞所消除，表明CCT参与新生IC-2的折叠。采用重组CCT复合物的体外翻译实验表明，CCT能够在从核糖体释放后结合新合成的IC-2，这与IC-2折叠中的作用一致。(C)2015 Elsevier Inc. All rights reserved.
Cytoplasmic dynein is a macromolecular motor complex with diverse function's in eukaryotic cells. Dynein plays essential roles in intracellular transport of organelles and mitosis, mediated in part by interactions between the dynein intermediate chain 2 (IC-2) subunits and adapter proteins that bind specific cargos. In experiments to identify phosphorylation-dependent binding partners for IC-2 we instead identified a phosphorylation-independent binding partner, the cytosolic chaperonin containing T complex protein 1 (CCT). CCT consists of eight subunits (CCT1-8) and facilitates folding of a subset of newly synthesized proteins. We confirmed interactions between IC-2 and CCT5 and CCT8 in co-immunoprecipitation experiments and determined that the C-terminal half of IC-2 is necessary and sufficient to bind CCT8. Interestingly, co-immunoiprecipitation of IC-2 and CCT is abolished by prior cycloheximide treatment of cells, suggesting that CCT participates in folding of nascent IC-2. In vitro translation experiments employing recombinant CCT complex demonstrated that CCT is able to bind newly synthesized IC-2 after release from the ribosome consistent with a role in folding of IC-2. (C) 2015 Elsevier Inc. All rights reserved.