Garrott Sharon R, Gillies John P, Siva Aravintha, Little Saffron R, Jbeily Rita Ei, DeSantis Morgan E
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
bioRxiv. 2023 Jan 25:2023.01.25.525437. doi: 10.1101/2023.01.25.525437.
Dynein is the primary minus-end-directed microtubule motor [1]. To achieve activation, dynein binds to the dynactin complex and an adaptor to form the "activated dynein complex" [2, 3]. The protein Lis1 aids activation by binding to dynein and promoting its association with dynactin and adaptor [4, 5]. Ndel1 and its orthologue Nde1 are dynein and Lis1 binding proteins that help control where dynein localizes within the cell [6]. Cell-based assays suggest that Ndel1/Nde1 also work with Lis1 to promote dynein activation, although the underlying mechanism is unclear [6]. Using purified proteins and quantitative binding assays, we found that Ndel1's C-terminal region contributes to binding to dynein 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 Ndel1's C-terminal domain 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. Our work suggests that , 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.
动力蛋白是主要的向微管负端移动的分子马达[1]。为实现激活，动力蛋白会与动力蛋白激活蛋白复合体及一个衔接蛋白结合，形成“激活的动力蛋白复合体”[2,3]。Lis1蛋白通过与动力蛋白结合并促进其与动力蛋白激活蛋白复合体及衔接蛋白的结合来协助激活过程[4,5]。Ndel1及其同源物Nde1是与动力蛋白和Lis1结合的蛋白，有助于控制动力蛋白在细胞内的定位[6]。基于细胞的实验表明，Ndel1/Nde1也与Lis1共同作用来促进动力蛋白的激活，尽管其潜在机制尚不清楚[6]。通过使用纯化蛋白和定量结合实验，我们发现Ndel1的C末端区域有助于与动力蛋白结合，并对与Lis1的结合起负调节作用。通过单分子成像和蛋白质生物化学方法，我们观察到Ndel1以两种不同方式抑制动力蛋白的激活。首先，Ndel1不利于激活的动力蛋白复合体的形成。我们发现Ndel1 C末端结构域中的模拟磷酸化突变增强了其抑制动力蛋白-动力蛋白激活蛋白复合体-衔接蛋白复合体形成的能力。其次，我们观察到Ndel1同时与动力蛋白和Lis1相互作用，并将Lis1从其与动力蛋白的结合位点上隔离出来。这样一来，Ndel1阻止了Lis1介导的动力蛋白激活。我们的研究表明，Ndel1是动力蛋白激活的负调节因子，这与细胞研究中Ndel1促进动力蛋白活性的结果形成对比。为使我们的研究结果与之前的工作相协调，我们推测Ndel1的作用是将动力蛋白和Lis1聚集在一起，同时使动力蛋白处于抑制状态。我们推测在细胞环境中可以触发Ndel1的释放，从而实现动力蛋白的定时激活。
