Mitochondrial Outer-membrane E3Ligase MUL1Regulates Selenite-induced Mitophagy And Ubiquitinates ULK1

Mitochondria serve as membrane sources and signaling platforms for regulating autophagy. However, accumulating evidence has shown that damaged mitochondria are removed through both selective mitophagy and general autophagy in response to mitochondrial and oxidative stresses. Protein ubiquitination through mitochondrial E3ligases plays an integrative role in mitochondrial outer membrane protein degradation, mitochondrial dynamics and mitophagy.In our study, we identified MUL1, a mitochondria-localized E3ligase regulates selenite-induced mitophagy in an ATG5and ULK1-dependent manner. Selenite was well-known for its chemopreventive effects through induction of apoptosis in cancer cells. We used low concentration of selenite as an effective stimulus of mitochondria specific autophagy. Our results showed that treatment of selenite lead to the significant accumulation of LC3II and degradation of several mitochondria residing proteins. Knockdown of endogenous expression of MUL1blocked the selenite induced mitophagy to a great extent compared to knockdown of other mitochondrial E3ligases, indicating MUL1as a potential mediator of the process. Conversely, ectopic expression of MUL1induced mitophagy in HeLa cells and mitochondrial fragmentation and the perinuclear distribution. Interestingly, our data showed that ULK1partially translocated to mitochondria and interacted with MUL1, leading to subsequent degradation through proteasome-dependent pathway. Furthermore, in vivo and in vitro ubiqtination experiment revealed that MUL1promoted the poly-ubiquitination of ULK1mainly via Lys48-linked ubiquitinatin chains. Thus, we identify ULK1as a novel substrate of MULl. These results suggest a new link of mitochondria with autophagy regulation and provide a new mechanism for the beneficial effects of selenium as a chemopreventive agent.