The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.

Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. RBX2, RING-Box Protein 2; SAG, Sensitive to Apoptosis Gene; Ub, Ubiquitin; pS65-Ub, phosphorylated Ub at serine 65; MAVS, mitochondrial antiviral-signaling protein; AAV, adeno-associated virus; AV, adenovirus; siRNA, Small interfering RNA; GFP, green fluorescent protein; CUL, cullin; RING, Really Interesting New Gene; CRLs, cullin-RING ligases; CSN, COP9 signalosome; APEX2, ascorbate peroxidase 2; mito, mitochondrial; cyto, cytosolic; MOM, mitochondrial outer membrane; CCCP, Carbonyl Cyanide Chlorophenylhydrazone; OMP25, Outer membrane protein 25; PK, proteinase K; HA, hemagglutinin; TMRM, Tetramethylrhodamine methyl ester perchlorate; αMHC,α-myosin heavy chain; CKO, cardiomyocyte-specific knockout; TAM, tamoxifen; TMT, tandem mass tag; KD, knockdown; CTL, control; MCM, MerCreMer; iCKO, inducible cardiomyocyte-specific knockout; BFA, bafilomycin A1; PCA, principle component analysis; MS, Mass spectrometry; DEPs, differentially expressed proteins; FC, fold change; FDR, False Discovery Rate; KEGG, Kyoto encyclopedia of genes and genomes; ER, endoplasmic reticulum; DKO, double knockout; CM, cardiomyocyte; cTnT, cardiac troponin T; NRVCs, neonatal rat ventricular cardiomyocytes; NRVMs, neonatal mouse ventricular cardiomyocytes; NMVFs, neonatal mouse ventricular fibroblasts; HF, heart failure; KO, knockout; MF, Molecular Functions; CC, Cellular Components; BP, Biological Process; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; SCF, Skp1-Cullin 1-F-box.