Abstract
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 pulldown 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 is 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 the 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.
Highlights
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)
Live-cell imaging and biochemical pulldown experiments revealed that p27 is a component of the dynactin complex in A. nidulans, it is dispensable for dynein– dynactin to interact with ⌬C-HookA and is not critical for dyneinmediated early endosome transport
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
Summary
P25 is known to be critical for early endosome transport (17), it has been unknown whether p27, the binding partner of p25 (4, 14, 18, 19), is involved in the same process. Mutants containing the ⌬C, ⌬L1, KYH, and N-GA mutations showed abnormal accumulation of early endosomes at the hyphal tip (Fig. 4, A and B). To determine whether the ⌬C, ⌬L1, KYH, and the N-AG mutations affect HookA binding, we introduced the ⌬CHookA-S allele into these mutants by genetic crosses and tested whether it can be pulled down with p25–GFP containing one of these mutations (Fig. 4D). The amounts of p150 pulled down were not significantly different from normal (Fig. 4, D and E) These results indicate that the defects in binding HookA do not result from the inability of the mutant p25 proteins to bind dynactin. Rather, these mutations affect the ability of p25 to mediate the HookA– dynein– dynactin interaction. Our results suggest that p25 enhances the dynactin–KinA interaction, and dynactin enhances the dynein–KinA interaction
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