Abstract
Dynamin-2 (Dyn2) is ubiquitously expressed and catalyzes membrane fission during clathrin-mediated endocytosis in nonneuronal cells. We have previously shown that Dyn2 inefficiently generates membrane curvature and only mediates fission of highly curved membranes. This led to the hypothesis that other endocytic accessory proteins (EAPs) generate curvature needed to sculpt a sufficiently narrow neck to trigger Dyn2 assembly and fission. Candidates for this activity are EAPs that bind to the dynamin proline/arginine-rich domain (PRD) through their SH3 (src homology-3) domains and also encode curvature-generating BAR (Bin/Amphiphysin/Rvs) domains. We show that at low concentrations, amphiphysin and endophilin, but not SNX9 or the curvature-generating epsin N-terminal homology (ENTH) domain, are able to generate tubules from planar membrane templates and to synergize with Dyn2ΔPRD to catalyze vesicle release. Unexpectedly, SH3-PRD interactions were inhibitory and reciprocally regulate scaffold assembly. Of the three proteins studied, only full-length amphiphysin functions synergistically with full-length Dyn2 to catalyze vesicle release. The differential activity of these proteins correlates with the relative potency of their positive, curvature-generating activity, and the negative regulatory effects mediated by SH3 domain interactions. Our findings reveal opportunities for the spatio-temporal coordination of membrane curvature generation, dynamin assembly, and fission during clathrin-mediated endocytosis.
Highlights
Membrane curvature generation is essential for dynamin-2-catalyzed membrane fission and vesicle release
Their curvature generating N-BAR domains synergize with Dyn2 to catalyze vesicle release, the SH3-proline/arginine-rich domain (PRD) interactions are inhibitory
The three dynamin binding partners we studied, amphiphyisin, endophilin, and sorting nexin 9 (SNX9), differentially effect the ability of Dyn2 to catalyze membrane fission, which presumably reflects differences in the relative potency of their positive, curvature generating activities and the negative regulatory effects mediated by SH3 domain interactions
Summary
Membrane curvature generation is essential for dynamin-2-catalyzed membrane fission and vesicle release. This led to the hypothesis that other endocytic accessory proteins (EAPs) generate curvature needed to sculpt a sufficiently narrow neck to trigger Dyn assembly and fission Candidates for this activity are EAPs that bind to the dynamin proline/arginine-rich domain (PRD) through their SH3 (src homology-3) domains and encode curvature-generating BAR (Bin/Amphiphysin/Rvs) domains. Dynamin-2 (Dyn2), the ubiquitously-expressed isoform, can assemble onto curved templates and catalyze fission, it is much less efficient than Dyn in generating membrane curvature and catalyzing vesicle release from planar membranes [9] These results suggest that other curvature-generating EAPs function upstream to generate the narrow neck needed to activate Dyn for CCV release. We have systematically studied the effects of BAR domain-containing dynamin binding partners, amphiphysin, endophilin, and SNX9, as well as epsin, on dynamin-catalyzed vesicle release from planar SUPER templates. Our results reveal complexity in the functional interplay between dynamin and its SH3 and BAR-domain containing partners that may reflect a hierarchy of interactions that combine to regulate curvature generation and dynamin activity in CME
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