RESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Endosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Transporte Biológico , Línea Celular , Endocitosis/genética , Células HEK293 , Células HeLa , Humanos , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Unión Proteica , Mapeo de Interacción de Proteínas , Transferrina/genética , Transferrina/metabolismo , Ubiquitinación , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
The peripheral protein quality control (QC) system removes non-native membrane proteins, including ΔF508-CFTR, the most common CFTR mutant in cystic fibrosis (CF), from the plasma membrane (PM) for lysosomal degradation by ubiquitination. It remains unclear how unfolded membrane proteins are recognized and targeted for ubiquitination and how they are removed from the apical PM. Using comprehensive siRNA screens, we identified RFFL, an E3 ubiquitin (Ub) ligase that directly and selectively recognizes unfolded ΔF508-CFTR through its disordered regions. RFFL retrieves the unfolded CFTR from the PM for lysosomal degradation by chaperone-independent K63-linked poly-ubiquitination. RFFL ablation enhanced the functional expression of cell-surface ΔF508-CFTR in the presence of folding corrector molecules, and this effect was further improved by inhibiting the Hsc70-dependent ubiquitination machinery. We propose that multiple peripheral QC mechanisms evolved to dispose of non-native PM proteins and to preserve cellular proteostasis, even at the cost of eliminating partially functional polypeptides.