RFFL Knockout Cell Line (HEK293)

The ubiquitin-proteasome system contributes to protein quality control, involving E3 ligases that ubiquitinate proteins and leading to their degradation. The dysregulation of protein degradation results in the abnormal accumulation of proteins and is implicated in the pathology of diverse diseases, making targeted protein degradation a promising therapeutic strategy. Here, we focus on RFFL, an endosome-associated RING E3 ligase involved in mitochondrial homeostasis and the clearance of misfolded cystic fibrosis transmembrane conductance regulator proteins. Using label-free quantitative mass spectrometry based proteomics for interactome and differential expression analyses, we systematically investigated and identified putative substrates of RFFL. For more confident identification, we performed these analyses on three cell lines that we generated: an RFFL knockout cell line generated using CRISPR/Cas9, another cell line rescuing RFFL expression when complemented with KO cells with stably expressing RFFL cDNA, and wild-type cells. We validated JMJD6 and DNAJB11 as substrates of endogenous RFFL, providing orthogonal validation and confidence in our screening approach. We demonstrated that RFFL ubiquitinates and degrades JMJD6 and DNAJB11 via the proteasomal pathway using in vivo assays. Interestingly, we also discovered a hitherto unknown role of RFFL in lipid metabolism. Collectively, this study provides the first comprehensive and unbiased analysis of RFFL substrates employing multiple complementary approaches.

Mitochondrial homeostasis is ensured through communication between diverse cellular organelles, including mitochondria, the endoplasmic reticulum (ER), lysosomes and endosomes. Although it is known that mitofusins regulate mitochondrial networks and ER contacts, their role in endosomal-mitochondrial interactions remains unclear. Previously, we have reported that vesicles positive for the endosomal ubiquitin ligase RFFL are associated with damaged mitochondria and prime the organelle for PRKN recruitment. Now, we establish that RFFL is a ubiquitin ligase for mitofusin 2 (MFN2). Using electron microscopy and confocal imaging analyses, we demonstrate that RFFL-knockout cells exhibit enlarged mitochondrial morphology. RFFL interacts at an endogenous level with MFN2 and contributes to its ubiquitylation upon mitochondrial damage. Recombinant RFFL interacts and ubiquitylates MFN2 protein in vitro. Furthermore, exogenous RFFL, in a ligase-dependent manner, specifically reduces the exogenous protein levels of both MFN1 and MFN2, but not that of DRP1, and also perturbs lipid homeostasis. Importantly, we show that the hyperfused mitochondria morphology reported with expression of pathogenic disease mutants of MFN2 (T206I and R364W) of Charcot-Marie-Tooth disease type 2A can be rescued by RFFL co-expression. The study unravels novel mechanisms involving endosomal ubiquitin ligases in mitochondrial networks.