Damaged mitochondria are selectively eliminated in a process called mitophagy. Parkin and PINK1, proteins mutated in Parkinson’s disease, amplify ubiquitin signals on damaged mitochondria with the subsequent activation of autophagic machinery. Autophagy adaptors are thought to link ubiquitinated mitochondria and autophagy through ATG8 protein binding. Here, we establish methods for inducing mitophagy by mitochondria-targeted ubiquitin chains and chemical-induced mitochondrial ubiquitination. Using these tools, we reveal that the ubiquitin signal is sufficient for mitophagy and that PINK1 and Parkin are unnecessary for autophagy activation per se. Furthermore, using phase-separated fluorescent foci, we show that the critical autophagy adaptor OPTN forms a complex with ATG9A vesicles. Disruption of OPTN–ATG9A interactions does not induce mitophagy. Therefore, in addition to binding ATG8 proteins, the critical autophagy adaptors also bind the autophagy core units that contribute to the formation of multivalent interactions in the de novo synthesis of autophagosomal membranes near ubiquitinated mitochondria.

Manipulation of protein stability with small molecules has a great potential for both basic research and clinical therapy. Recently, we have developed a series of hybrid small molecules named SNIPER (Specific and Non‐genetic IAP‐dependent Protein ER aser) that induces degradation of target proteins via ubiquitin‐proteasome system. Here we report the activities of SNIPER(ER) that targets estrogen receptor alpha (ERα) for degradation. SNIPER(ER) induced degradation of ERα and inhibited estrogen‐dependent expression of pS2 gene in an estrogen‐dependent breast cancer cell line MCF‐7. A proteasome inhibitor MG132 and siRNA‐mediated downregulation of cIAP1 abrogated the SNIPER(ER)‐induced ERα degradation, suggesting that the ERα is degraded by proteasome subsequent to cIAP1‐mediated ubiquitylation. Intriguingly, after the ERα degradation, the SNIPER(ER)‐treated MCF‐7 cells undergo rapid cell death. Detailed analysis indicated that SNIPER(ER) caused necrotic cell death accompanied by a release of HMGB1, a marker of necrosis, from the cells. Following the ERα degradation, reactive oxygen species (ROS) was produced in the SNIPER(ER)‐treated MCF‐7 cells, and an anti‐oxidant N‐acetylcysteine inhibited the necrotic cell death. These results indicate that SNIPER(ER) induces ERα degradation, ROS production and necrotic cell death, implying a therapeutic potential of SNIPER(ER) as a lead for the treatment of ERα‐positive breast cancers.