Cytoplasmic dynein binds its cargoes via the dynactin complex and cargo adapters， and the dynactin pointed-end protein p25 is required for dynein-dynactin binding to the early endosomal dynein adapter HookA (Hook in the fungus Aspergillus nidulans). However， it is unclear whether the HookA-dynein-dynactin interaction requires p27， another pointed-end protein forming heterodimers with p25 within vertebrate dynactin. Here， live-cell imaging and biochemical pull-down experiments revealed that although p27 is a component of the dynactin complex in A. nidulans， it is dispensable for dynein-dynactin to interact with ΔC-HookA (cytosolic HookA lacking its early endosome-binding C terminus) and not critical f... More
Cytoplasmic dynein binds its cargoes via the dynactin complex and cargo adapters， and the dynactin pointed-end protein p25 is required for dynein-dynactin binding to the early endosomal dynein adapter HookA (Hook in the fungus Aspergillus nidulans). However， it is unclear whether the HookA-dynein-dynactin interaction requires p27， another pointed-end protein forming heterodimers with p25 within vertebrate dynactin. Here， live-cell imaging and biochemical pull-down experiments revealed that although p27 is a component of the dynactin complex in A. nidulans， it is dispensable for dynein-dynactin to interact with ΔC-HookA (cytosolic HookA lacking its early endosome-binding C terminus) and not critical for dynein-mediated early endosome transport. Using mutagenesis， imaging， and biochemical approaches， we found that several p25 regions are required for the ΔC-HookA-dynein-dynactin interaction， with the N terminus and Loop1 being the most critical regions. Interestingly， p25 was also important for the microtubule (MT) plus-end accumulation of dynactin. This p25 function in dynactin localization also involved p25's N terminus and Loop1 critical for the ΔC-HookA-dynein-dynactin interaction. Given that dynactin's MT plus-end localization does not require HookA and that the kinesin-1-dependent plus-end accumulation of dynactin is unnecessary for the ΔC-HookA-dynein-dynactin interaction， our results indicate that p25 plays a dual role in cargo binding and dynactin regulation. As cargo adapters are implicated in dynein activation via binding to dynactin's pointed end to switch the conformation of p150， a major dynactin component， our results suggest p25 as a critical pointed-end protein involved in this process.
