The integral function of the endocytic recycling compartment is regulated by RFFL-mediated ubiquitylation of Rab11 effectors

Endocytic trafficking is regulated by ubiquitination of cargoes and endocytic machineries. The role of ubiquitination in lysosomal delivery has been well documented, but that in the recycling pathway is largely unknown. Here we report that ubiquitin (Ub) ligase RFFL regulates ubiquitination of endocytic recycling regulators. RFFL dominant-negative (DN) mutant induced clustered endocytic recycling compartments (ERC) and delayed endocytic cargo recycling without affecting lysosomal traffic. BioID RFFL interactome analysis revealed that RFFL interacts with Rab11 effectors EHD1, MICALL1 and class I Rab11-FIPs. The RFFL DN mutant strongly captured these Rab11 effectors and inhibited their ubiquitination. The prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC and to delay cargo recycling. RFFL directly ubiquitinates these Rab11 effectors in vitro , but RFFL KO reduced only ubiquitination of Rab11-FIP1. RFFL KO had a minimal effect on the ubiquitination of EHD1, MICALL1, and Rab11-FIP2, and failed to delay transferrin recycling. These results suggest that multiple Ub ligases including RFFL regulate the ubiquitination of Rab11 effectors, determining the integral function of the ERC.