A Regulatory Signaling Loop Controls FUNDC1-mediated Mitophagy

Mitochondria function as a powerhouse for energy production, which is essential for cell survival. They also play central roles for intracellular free radical production and programmed cell death. It is thus important for a cell to maintain a proper mitochondrial quality and quantity. During the evolution, cell has developed elaborate systems to monitor mitochondrial quality. In particular, redundant and/or damaged mitochondria can be selectively removed by the mechanism of mitochondrial autophagy, or mitophagy. As dysregulation of mitophagy was closely associated with a number of diseases, it attracts great attention across biomedical research fields in addition to mitochondrial and autophagy research. Several mechanisms were proposed to be involved in selective mitophagy; however, the molecular details of how damaged mitochondria are recognized for disposal are not clear.FUNDCl is a newly identified mitophagy receptor which is a three transmembrane protein localizing at outer mitochondrial membrane, with its N terminal exposing to cytosol. FUNDC1is a highly conserved and ubiquitously expressed protein. It possesses a classic LC3-interacting region YxxL (LIR) which interacts with LC3and recruits isolation membrane to engulf mitochondria leading to mitophagy upon mitophagy stresses. In this study, we explored the molecular regulation of FUNDC1-mediated mitophagy.We have found that reversible phosphorylation of FUNDC1is critical for receptor-mediated mitophagy. Tyrl8in the LIR of FUNDC1and Ser13near the LIR are highly phosphorylated in normal unstressed condition and are dephosphorylated upon mitophagy stresses. Dephosphorylation enhances the interaction between FUNDC1and LC3, thus the activation of mitophagy. Biochemical analysis revealed that phosphorylated FUNDCl has weak binding affinity to LC3. Unphosphorylated FUNDC1peptide encompassing the Ser13and LIR of FUNDCl competitively blocks the interaction between endogenous FUNDC1and LC3to a greater extent than the phosphorylated form. Cell permeable unphosphorylated peptide prevented mitochondrial protein degradation in a dose-dependent manner.To understand the molecular mechanisms of the reversible phosphorylation of FUNDC1, we have identified that mitochondrially localized PGAM5is the phosphatase responsible for the dephosphorylation of FUNDC1at Ser13and mitochondrial stresses increase the binding ability of PGAM5to FUNDC1. Knockdown of PGAM5inhibits FUNDC1dephosphorylation and mitophagy, which can be rescued by re-introduction of wild type PGAM5, but not the phosphatase dead mutant. Also, over-expressing S13A mutant FUNDC1which could not be phosphorylated, but not wild type FUNDC1, can restore the attenuated mitophagy in PGAM5knockdown cells.We have also addressed the question which kinases are responsible for maintaining the phosphorylation status of FUNDCl. We have identified that CK2and Src are the kinases responsible for Ser13and Tyr18phosphorylation, respectively. These two kinases are able to phosphorylate Ser13and Tyr18and inhibit the interaction between FUNDC1and LC3and subsequent mitophagy.We showed that the phosphorylation status of Serl3and Tyr18may cooperate to regulate FUNDC1-mediated mitophagy. Mitochondrial stresses decrease both interactions between FUNDC1and CK2and Src kinases. Treating cells with CK2inhibitor or Src inhibitor alone fails to induce mitophagy although dephosphorylation at respective site occurs. Interestingly, application of both inhibitors will trigger mitophagy.In summary, in normal condition, FUNDC1is phosphorylated at Ser13by CK2and Tyr18by Src to inhibit its interaction with LC3and mitophagy. Upon mitophagy stresses, CK2and Src will be released from and PGAM5will bind to FUNDC1, leading to FUNDCl dephosphorylation which will endow FUNDC1with enhanced binding affinity with LC3for the activation of mitophagy. Our research provides a mechanism through which reversible phosphorylation regulates FUNDC1-mediated mitophagy and offers new insight to understand the physiological function of mitophagy.