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

Abstract

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.

Cytomegalovirus Generates Assembly Compartment in the Early Phase of Infection by Perturbation of Host-Cell Factors Recruitment at the Early Endosome/Endosomal Recycling Compartment/Trans-Golgi Interface

“…The molecular rheostat function based on the fine titration of the ubiquitination by the cooperative action of a ubiquitin (Ub) ligase and a deubiquitinating enzyme (DUB) has been demonstrated in the actin-assembly function of the WASH complex at EEs (Hao et al, 2015). A recent study demonstrated that ubiquitination by multiple Ub ligases and interaction with several DUBs also regulates ERC dynamics (Sakai et al, 2019). Inhibition of an Ub ligase that acts at the ERC (Sakai et al, 2019) or its over-expression (Coumailleau et al, 2004) impairs the segregation of EEs and the ERC, inhibits ERC-to-TGN trafficking, and redistributes a TGN resident protein (TGN46) into the ERC.…”

“…A recent study demonstrated that ubiquitination by multiple Ub ligases and interaction with several DUBs also regulates ERC dynamics (Sakai et al, 2019). Inhibition of an Ub ligase that acts at the ERC (Sakai et al, 2019) or its over-expression (Coumailleau et al, 2004) impairs the segregation of EEs and the ERC, inhibits ERC-to-TGN trafficking, and redistributes a TGN resident protein (TGN46) into the ERC. Spatiotemporal regulation of ubiquitination of host-cell factors that control endosomal tubulation (i.e., EHD1 and MICAL-L1) has been proposed to be essential for segregation of EE and the ERC (Sakai et al, 2019).…”

“…Inhibition of an Ub ligase that acts at the ERC (Sakai et al, 2019) or its over-expression (Coumailleau et al, 2004) impairs the segregation of EEs and the ERC, inhibits ERC-to-TGN trafficking, and redistributes a TGN resident protein (TGN46) into the ERC. Spatiotemporal regulation of ubiquitination of host-cell factors that control endosomal tubulation (i.e., EHD1 and MICAL-L1) has been proposed to be essential for segregation of EE and the ERC (Sakai et al, 2019). Thus, the membrane flow at the ERC interface could be regulated by Ub-based molecular rheostats, and CMVs may reshape the EE-ERC-TGN interface by dysregulation of the coordinated function of ERC-associated Ub ligases and DUBs.…”

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitylation as a Novel Pharmaceutical Target for Cystic Fibrosis

“…RFFL has a Fab-1, YGL023, Vps27, and EEA1 (FYVE)-like domain that binds to phosphatidylinositol (PI) 3-phosphate (PI(3)P) and PI(5)P in the N-terminal region, the Really Interesting New Gene (RING) domain which is important for ubiquitin ligase activity in the C-terminal region, and multiple disordered regions within other regions [48]. RFFL does not have a transmembrane domain, but its N-terminal domain is palmitoylated and is localized on the cytoplasmic side of the plasma and endosomal membranes [49,50]. RFFL directly recognizes the NBD1 of CFTR through the N-terminal disordered regions.…”

Activated Protein Kinase C (PKC) Is Persistently Trafficked with Epidermal Growth Factor (EGF) Receptor

“…Of the other proteins present, Rab11 is considered a marker but is also present in early endosomes and multivesicular bodies (review [21,25,26]). The early endosomal markers, Rab5 and EEA1, and late endosomal markers, Rab7 and Lamp1, accumulate in the ERC [27]. The significance of our findings is that one conformation of PKC, thought to include the enzymatically active molecules, is processed to another during endosomal trafficking.…”