Gp78-mediated basal mitophagy promotes mitochondrial health and limits mitochondrial ROS production

Mitochondria are major sources of cytotoxic reactive oxygen species (ROS) that contribute to cancer progression. Mitophagy, the selective elimination of mitochondria by autophagy, monitors and maintains mitochondrial health and integrity, eliminating ROS-producing mitochondria. However, mechanisms underlying mitophagic control of mitochondrial homeostasis under basal conditions remain poorly understood. Gp78 E3 ubiquitin ligase is an endoplasmic reticulum membrane protein that induces mitochondrial fission and mitophagy of depolarized mitochondria. Here, we report that CRISPR/Cas9 knockout of Gp78 in HT-1080 fibrosarcoma cells increased mitochondrial volume and rendered cells resistant to carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitophagy. These effects were phenocopied by knockdown of the essential autophagy protein ATG5 in wild-type HT-1080 cells, suggesting that endogenous Gp78 promotes elimination of mitochondria by autophagy in wild-type HT-1080 cells. Application of a spot detection algorithm (SPECHT) to GFP-mRFP tandem fluorescent-tagged LC3 (tfLC3) transfected cells showed increased accumulation of GFP-mRFP-positive autophagic vacuoles upon treatment with Bafilomycin A under basal conditions and in response to CCCP. Gp78 knockout and inhibition of basal autophagic flux by ATG5 knockdown resulted in reduced mitochondrial potential and increased mitochondrial ROS. Live cell analysis of tfLC3 in HT-1080 cells showed the preferential association of autophagosomes with mitochondria of reduced potential. Gp78-dependent basal mitophagic flux therefore promotes mitochondrial health and reduces ROS production in cancer cells.