Ndel1 disfavors dynein-dynactin-adaptor complex formation in two distinct ways.
Ndel1 disfavors dynein-dynactin-adaptor complex formation in two distinct ways.
复制标题
DOI：
10.1016/j.jbc.2023.104735
复制
发表时间：
2023-06
影响因子：
4.8
通讯作者：
DeSantis, Morgan E
中科院分区：
文献类型：
--
作者：
Garrott, Sharon R;Gillies, John P;Siva, Aravintha;Little, Saffron R;El Jbeily, Rita;DeSantis, Morgan E
动力蛋白是主要的负端定向微管运动蛋白。为了实现激活，动力蛋白与动力蛋白复合物和一个接头结合，形成“活化的动力蛋白复合物”。Lis1蛋白通过与动力蛋白结合并促进其与动力蛋白和接头的结合来辅助激活。Ndel1及其类似物Nde1是动力蛋白和lis1结合蛋白，帮助控制动力蛋白在细胞内的定位。基于细胞的实验表明，Ndel1-Nde1也与Lis1一起促进动力蛋白的激活，尽管其潜在机制尚不清楚。通过纯化蛋白和定量结合实验，我们发现Ndel1的c端区域有助于动力蛋白的结合，并负调控与Lis1的结合。通过单分子成像和蛋白质生物化学，我们观察到Ndel1以两种不同的方式抑制动力蛋白的激活。首先，Ndel1不利于激活动力蛋白复合物的形成。我们发现Ndel1 c端结构域的拟磷突变增加了其抑制动力蛋白-动力蛋白-适配器复合物形成的能力。其次，我们观察到Ndel1同时与动力蛋白和Lis1相互作用，并将Lis1从动力蛋白结合位点分离。在此过程中，Ndel1阻止了lis1介导的动力蛋白激活。总之，我们的工作表明，在体外，Ndel1是动力蛋白激活的负调节因子，这与细胞研究中Ndel1促进动力蛋白活性形成对比。为了使我们的发现与之前的工作相一致，我们假设Ndel1的功能是将动力蛋白和Lis1结合在一起，同时使动力蛋白处于抑制状态。我们推测Ndel1的释放可以在细胞设置中触发，以允许定时动力蛋白激活。
Dynein is the primary minus-end-directed microtubule motor protein. To achieve activation, dynein binds to the dynactin complex and an adaptor to form the “activated dynein complex.” The protein Lis1 aids activation by binding to dynein and promoting its association with dynactin and the adaptor. Ndel1 and its paralog Nde1 are dynein- and Lis1-binding proteins that help control dynein localization within the cell. Cell-based assays suggest that Ndel1–Nde1 also work with Lis1 to promote dynein activation, although the underlying mechanism is unclear. Using purified proteins and quantitative binding assays, here we found that the C-terminal region of Ndel1 contributes to dynein binding and negatively regulates binding to Lis1. Using single-molecule imaging and protein biochemistry, we observed that Ndel1 inhibits dynein activation in two distinct ways. First, Ndel1 disfavors the formation of the activated dynein complex. We found that phosphomimetic mutations in the C-terminal domain of Ndel1 increase its ability to inhibit dynein–dynactin–adaptor complex formation. Second, we observed that Ndel1 interacts with dynein and Lis1 simultaneously and sequesters Lis1 away from its dynein-binding site. In doing this, Ndel1 prevents Lis1-mediated dynein activation. Together, our work suggests that in vitro, Ndel1 is a negative regulator of dynein activation, which contrasts with cellular studies where Ndel1 promotes dynein activity. To reconcile our findings with previous work, we posit that Ndel1 functions to scaffold dynein and Lis1 together while keeping dynein in an inhibited state. We speculate that Ndel1 release can be triggered in cellular settings to allow for timed dynein activation.