Activation of Nedd4L Ubiquitin Ligase by FCHO2-generated Membrane Curvature
Activation of Nedd4L Ubiquitin Ligase by FCHO2-generated Membrane Curvature
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FCHO2 生成的膜曲率激活 Nedd4L 泛素连接酶
DOI：
10.1101/2022.02.21.481372
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Nakanishi Hiroyuki
中科院分区：
文献类型：
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作者：
Sakamoto Yasuhisa;Uezu Akiyoshi;Kikuchi Koji;Suetsugu Shiro;Nakanishi Hiroyuki
C2-WW-HECT结构域泛素连接酶Nedd 4L通过介导货物分子（例如上皮钠通道（ENaC））的泛素化来调节内吞作用中的分选。ENaC泛素化缺陷导致Liddle综合征，一种遗传性高血压。Nedd 4L通过C2和HECT结构域之间的分子内相互作用而被催化自抑制，但其激活机制知之甚少。在这里，我们表明，Nedd 4L被FCHO 2（一种调节内吞作用的Bin-Amphiphysin-Rsv（BAR）结构域蛋白）雕刻的膜激活。我们发现FCHO 2是Nedd 4L介导的ENaC泛素化和内吞所必需的。Nedd 4L与FCHO 2共定位在网格蛋白包被的凹坑中，在那里它可能被激活。Nedd 4L被细胞中外源表达的FCHO 2 BAR结构域特异性募集并激活。此外，我们在体外重建FCHO 2诱导的Nedd 4L募集和激活。募集和激活都是由膜弯曲而不是蛋白质-蛋白质相互作用介导的。Nedd 4L C2结构域识别由FCHO 2 BAR结构域产生的特定程度的膜弯曲。因此，这种弯曲通过缓解自抑制来激活Nedd 4L。因此，我们首次展示了一种特定的功能（即，调节货物分选的酶的募集和活化）。
The C2-WW-HECT domain ubiquitin ligase Nedd4L regulates sorting in endocytosis by mediating ubiquitination of cargo molecules, such as the epithelial sodium channel (ENaC),. Defects in ENaC ubiquitination cause Liddle syndrome, a hereditary hypertension. Nedd4L is catalytically autoinhibited by an intramolecular interaction between the C2 and HECT domains, but the activation mechanism is poorly understood. Here, we show that Nedd4L is activated by membranes sculpted by FCHO2, a Bin-Amphiphysin-Rsv (BAR) domain protein that regulates endocytosis. We found that FCHO2 was required for Nedd4L-mediated ubiquitination and endocytosis of ENaC. Nedd4L co-localized with FCHO2 at clathrin-coated pits where it likely became activated. Nedd4L was specifically recruited to and activated by the FCHO2 BAR domain exogenously expressed in cells. Furthermore, we reconstitutedin vitroFCHO2-induced recruitment and activation of Nedd4L. 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. Consequently, this curvature activated Nedd4L by relieving autoinhibition. Thus, we show for the first time a specific functionality (i.e., recruitment and activation of an enzyme regulating cargo sorting) of membrane curvature by a BAR domain protein.