Processive transport by the microtubule motor cytoplasmic dynein requires the regulated assembly of a dynein-dynactin-adapter complex. Interactions between dynein and dynactin were initially ascribed to the dynein intermediate chain N-terminus and the dynactin subunit p150^Glued. However, recent cryo-EM structures have not resolved this interaction, questioning its importance. The intermediate chain also interacts with Nde1/Ndel1, which compete with p150^Glued for binding. We reveal that the intermediate chain N-terminus is a critical evolutionarily conserved hub that interacts with dynactin and Ndel1, the latter of which recruits LIS1 to drive complex assembly. In additon to revealing that the intermediate chain N-terminus is likely bound to p150^Glued in active transport complexes, our data support a model whereby Ndel1-LIS1 must dissociate prior to LIS1 being handed off to dynein in temporally discrete steps. Our work reveals previously unknown steps in the dynein activation pathway, and provide insight into the integrated activities of LIS1/Ndel1 and dynactin/cargo-adapters.

微管运动细胞质动力蛋白的过程运输需要动力蛋白-动力蛋白-适配器复合物的受调节组装。动力蛋白和动力蛋白之间的相互作用最初归因于动力蛋白中间链 N 末端和动力蛋白亚基 p150^胶合。然而，最近的冷冻电镜结构尚未解决这种相互作用，对其重要性提出质疑。中间链还与 Nde1/Ndel1 相互作用，后者与 p150 ^Glued竞争结合。我们揭示了中间链 N 末端是一个关键的进化保守中心，它与 dynactin 和 Ndel1 相互作用，后者招募 LIS1 来驱动复杂的组装。除了揭示中间链 N 末端可能与主动转运^复合物中的 p150 结合外，我们的数据还支持一个模型，即 Ndel1-LIS1 必须在 LIS1 以时间离散步骤传递给动力蛋白之前解离。我们的工作揭示了动力蛋白激活途径中以前未知的步骤，并提供了对 LIS1/Ndel1 和动力蛋白/货物适配器的整合活性的深入了解。