The Nedd4L ubiquitin ligase is activated by FCHO2-generated membrane curvature

The C2-WW-HECT domain ubiquitin ligase Nedd4L regulates membrane sorting during endocytosis through the ubiquitination of cargo molecules such as the epithelial sodium channel (ENaC). Nedd4L is catalytically autoinhibited by an intramolecular interaction between its C2 and HECT domains, but the protein's activation mechanism is poorly understood. Here, we show that Nedd4L activation is linked to membrane shape by FCHO2, a Bin-Amphiphysin-Rsv (BAR) domain protein that regulates endocytosis. FCHO2 was required for the Nedd4L-mediated ubiquitination and endocytosis of ENaC, with Nedd4L co-localizing with FCHO2 at clathrin-coated pits. In cells, Nedd4L was specifically recruited to, and activated by, the FCHO2 BAR domain. Furthermore, we reconstituted FCHO2-induced recruitment and activation of Nedd4L in vitro. Both the recruitment and activation were mediated by membrane curvature rather than protein-protein interactions. The Nedd4L C2 domain recognized a specific degree of membrane curvature that was generated by the FCHO2 BAR domain, with this curvature directly activating Nedd4L by relieving its autoinhibition. Thus, we show for the first time a specific function (i.e., recruitment and activation of an enzyme regulating cargo sorting) of membrane curvature by a BAR domain protein. Nedd4L is catalytically autoinhibited by an intramolecular interaction between the C2 and HECT domains. This work describes how the C2 domain recognizes FCHO2-generated membrane curvature, activating Nedd4L by relieving autoinhibition.FCHO2 is required for Nedd4L-mediated ubiquitination and endocytosis of the epithelial sodium channel.Nedd4L co-localizes with FCHO2 at clathrin-coated pits.Nedd4L is specifically recruited to, and activated by, the FCHO2 BAR domain.The recruitment and activation of Nedd4L is mediated by membrane curvature rather than protein-protein interactions. Binding to a specific degree of membrane curvature rather than protein-protein interactions relieves the autoinhibition of the Nedd4L ubiquitin ligase