The integral function of the endocytic recycling compartment is regulated by RFFL-mediated ubiquitylation of Rab11 effectors
The integral function of the endocytic recycling compartment is regulated by RFFL-mediated ubiquitylation of Rab11 effectors
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DOI：
10.1242/jcs.228007
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发表时间：
2019-02-01
影响因子：
4
通讯作者：
Okiyoneda, Tsukasa
中科院分区：
文献类型：
--
作者：
Sakai, Ryohei;Fukuda, Ryosuke;Okiyoneda, Tsukasa
内吞运输受货物和内吞机制的泛素化（也称为泛素化）调节。泛素化在溶酶体传递中的作用已被充分记录，但其在再循环途径中的作用在很大程度上是未知的。在这里，我们报告说，泛素（Ub）连接酶RFFL调节内吞再循环调节泛素化。RFFL显性阴性（DN）突变体诱导内吞再循环区室（ERCs）的聚集和延迟内吞货物再循环，而不影响溶酶体运输。BioID RFFL相互作用组分析显示，RFFL与Rab 11效应子EHD 1、MICALL 1和I类Rab 11-FIP相互作用。RFFL DN突变体强烈捕获这些Rab 11效应子并抑制其泛素化。RFFL与Rab 11效应子的长期相互作用足以诱导成簇的ERC表型并延迟货物再循环。RFFL在体外直接泛素化这些Rab 11效应物，但RFFL敲除（KO）仅减少Rab 11-FIP 1的泛素化。RFFL KO对EHD 1、MICALL 1和Rab 11-FIP 2的泛素化作用最小，并且未能延迟转铁蛋白再循环。这些结果表明，包括RFFL在内的多种Ub连接酶调节Rab 11效应物的泛素化，决定了ERC的整体功能。
Endocytic trafficking is regulated by ubiquitylation (also known as ubiquitination) of cargoes and endocytic machineries. The role of ubiquitylation in lysosomal delivery has been well documented, but its role in the recycling pathway is largely unknown. Here, we report that the ubiquitin (Ub) ligase RFFL regulates ubiquitylation of endocytic recycling regulators. An RFFL dominant-negative (DN) mutant induced clustering of endocytic recycling compartments (ERCs) and delayed endocytic cargo recycling without affecting lysosomal traffic. A BioID RFFL interactome analysis revealed that RFFL interacts with the Rab11 effectors EHD1, MICALL1 and class I Rab11-FIPs. The RFFL DN mutant strongly captured these Rab11 effectors and inhibited their ubiquitylation. The prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC phenotype and to delay cargo recycling. RFFL directly ubiquitylates these Rab11 effectors in vitro, but RFFL knockout (KO) only reduced the ubiquitylation of Rab11-FIP1. RFFL KO had a minimal effect on the ubiquitylation of EHD1, MICALL1, and Rab11-FIP2, and failed to delay transferrin recycling. These results suggest that multiple Ub ligases including RFFL regulate the ubiquitylation of Rab11 effectors, determining the integral function of the ERC.