Sakamoto Yasuhisa, Uezu Akiyoshi, Kikuchi Koji, Kang Jangmi, Fujii Eiko, Moroishi Toshiro, Suetsugu Shiro, Nakanishi Hiroyuki
Department of Molecular Pharmacology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjyo, Kumamoto, 860-8556, Japan.
Faculty of Clinical Nutrition and Dietetics, Konan Women's University, 6-2-23 Morikita-machi, Kobe, 658-0001, Japan.
EMBO J. 2024 Dec;43(23):5883-5909. doi: 10.1038/s44318-024-00268-1. Epub 2024 Oct 14.
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.
C2-WW-HECT结构域泛素连接酶Nedd4L通过对货物分子（如上皮钠通道ENaC）进行泛素化作用来调节内吞作用过程中的膜分选。Nedd4L通过其C2和HECT结构域之间的分子内相互作用而发生催化性自抑制，但其蛋白激活机制仍知之甚少。在此，我们表明Nedd4L的激活与FCHO2（一种调节内吞作用的Bin-双栖蛋白-Rsv，即BAR结构域蛋白）所介导的膜形状有关。FCHO2是ENaC的Nedd4L介导的泛素化作用和内吞作用所必需的，Nedd4L与FCHO2在网格蛋白包被小窝处共定位。在细胞中，Nedd4L被特异性募集到FCHO2的BAR结构域并被其激活。此外，我们在体外重建了FCHO2诱导的Nedd4L的募集和激活过程。募集和激活均由膜曲率介导，而非蛋白质-蛋白质相互作用。Nedd4L的C2结构域识别由FCHO2的BAR结构域产生的特定程度的膜曲率，这种曲率通过解除其自抑制作用而直接激活Nedd4L。因此，我们首次展示了BAR结构域蛋白介导的膜曲率的一种特定功能（即募集和激活一种调节货物分选的酶）。
