Okada Kyoko, Iyer Bharat R, Lammers Lindsay G, Gutierrez Pedro, Li Wenzhe, Markus Steven M, McKenney Richard J
Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA, USA.
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA.
bioRxiv. 2023 Jan 14:2023.01.13.523097. doi: 10.1101/2023.01.13.523097.
Cytoplasmic dynein, the primary retrograde microtubule transport motor within cells, must be activated for processive motility through the regulated assembly of a dynein-dynactin-adapter (DDA) complex. The interaction between dynein and dynactin was initially ascribed to the N-terminus of the dynein intermediate chain (IC) and a coiled-coil of the dynactin subunit p150 . However, cryo-EM structures of DDA complexes have not resolve these regions of the IC and p150 , raising questions about the importance of this interaction. The IC N-terminus (ICN) also interacts with the dynein regulators Nde1/Ndel1, which compete with p150 for binding to ICN. Using a combination of approaches, we reveal that the ICN plays critical, evolutionarily conserved roles in DDA assembly by interacting with dynactin and Ndel1, the latter of which recruits the DDA assembly factor LIS1 to the dynein complex. In contrast to prior models, we find that LIS1 cannot simultaneously bind to Ndel1 and dynein, indicating that LIS1 must be handed off from Ndel1 to dynein in temporally discrete steps. Whereas exogenous Ndel1 or p150 disrupts DDA complex assembly , neither perturbs preassembled DDA complexes, indicating that the IC is stably bound to p150 within activated DDA complexes. Our study reveals previously unknown regulatory steps in the dynein activation pathway, and provides a more complete model for how the activities of LIS1/Ndel1 and dynactin/cargo-adapters are integrated to regulate dynein motor activity.
胞质动力蛋白是细胞内主要的逆行微管运输马达，必须通过动力蛋白-动力肌动蛋白-衔接蛋白（DDA）复合物的有序组装来激活，以实现持续运动。动力蛋白与动力肌动蛋白之间的相互作用最初被认为是动力蛋白中间链（IC）的N端与动力肌动蛋白亚基p150的卷曲螺旋之间的相互作用。然而，DDA复合物的冷冻电镜结构尚未解析IC和p150的这些区域，这引发了关于这种相互作用重要性的疑问。IC的N端（ICN）还与动力蛋白调节因子Nde1/Ndel1相互作用，后者与p150竞争结合ICN。通过多种方法相结合，我们发现ICN通过与动力肌动蛋白和Ndel1相互作用，在DDA组装中发挥关键的、进化上保守的作用，其中Ndel1将DDA组装因子LIS1招募到动力蛋白复合物中。与先前的模型不同，我们发现LIS1不能同时结合Ndel1和动力蛋白，这表明LIS1必须在时间上离散的步骤中从Ndel1传递给动力蛋白。虽然外源性Ndel1或p150会破坏DDA复合物的组装，但两者都不会干扰预先组装好的DDA复合物，这表明在活化的DDA复合物中IC与p150稳定结合。我们的研究揭示了动力蛋白激活途径中以前未知的调节步骤，并为LIS1/Ndel1和动力肌动蛋白/货物衔接蛋白的活性如何整合以调节动力蛋白马达活性提供了一个更完整的模型。
