Identification of a novel region of the cytoplasmic dynein intermediate chain important for dimerization in the absence of the light chains
Identification of a novel region of the cytoplasmic dynein intermediate chain important for dimerization in the absence of the light chains
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DOI：
10.1074/jbc.m511721200
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发表时间：
2006-04-07
影响因子：
4.8
通讯作者：
Pfister, KK
中科院分区：
文献类型：
--
作者：
Lo, KWH;Kan, HM;Pfister, KK
细胞质动力蛋白是一种多亚基蛋白质复合物，负责许多基于微管的细胞内运动。其货物结合域由五个亚基的二聚体组成：中间链、轻中间链、Tctex1、Roadblock 和 LC8 轻链。中间链在动力蛋白复合物中起着关键作用。它们结合三个轻链和包含运动域的重链，但人们对两条中间链如何相互作用知之甚少。有六种中间链亚型，并且假设不同的亚型可以调节特定的动力蛋白功能。然而，关于动力蛋白复合物中中间链亚型的潜在组合的数据很少。我们使用免疫共沉淀分析来证明六个中间链的同二聚体和异二聚体的所有组合都是可能的。因此，具有不同异构体组合的动力蛋白复合物的形成不受各种中间链之间的相互作用的限制。我们进一步试图确定中间链二聚化所需的结构域。对一系列截短和缺失突变体的分析表明，61 个氨基酸区域是中间链二聚化所必需的。该区域不包括 N 端卷曲螺旋、C 端 WD 重复结构域或 Tctex1、LC8 和 Roadblock 轻链的三个不同结合位点。纯化的重组多肽的分析凝胶过滤和共价交联进一步证明中间链可以在轻链不存在的情况下在体外二聚化。
Cytoplasmic dynein is the multisubunit protein complex responsible for many microtubule-based intracellular movements. Its cargo binding domain consists of dimers of five subunits: the intermediate chains, the light intermediate chains, and the Tctex1, Roadblock, and LC8 light chains. The intermediate chains have a key role in the dynein complex. They bind the three light chains and the heavy chains, which contain the motor domains, but little is known about how the two intermediate chains interact. There are six intermediate chain isoforms, and it has been hypothesized that different isoforms may regulate specific dynein functions. However, there are little data on the potential combinations of the intermediate chain isoforms in the dynein complexes. We used co-immunoprecipitation analyses to demonstrate that all combinations of homo- and heterodimers of the six intermediate chains are possible. Therefore the formation of dynein complexes with different combinations of isoforms is not limited by interaction between the various intermediate chains. We further sought to identify the domain necessary for the dimerization of the intermediate chains. Analysis of a series of truncation and deletion mutants showed that a 61-amino-acid region is necessary for dimerization of the intermediate chain. This region does not include the N-terminal coiled-coil, the C-terminal WD repeat domain, or the three different binding sites for the Tctex1, LC8, and Roadblock light chains. Analytical gel filtration and covalent cross-linking of purified recombinant polypeptides further demonstrated that the intermediate chains can dimerize in vitro in the absence of the light chains.