Dual-faced PUMA in CRC: A cytoplasmic autophagy repressor and mitochondrial mitophagy promoter

As Bcl-2 family members, PUMA and Bcl-X_L played critical roles in mitochondrial apoptosis. However, whether they can regulate autophagy, especially mitophagy, is not understood at all. In this study, we explore the interaction among PUMA and Bcl-X_L in different subcellular localizations, and their functions in autophagy and mitophagy respectively. The detailed mechanisms were determined by mitochondria purification, Co-IP, and western blot analysis. Moreover, living cell imaging was performed to determine the occurrence of mitophagy. We found that PUMA inhibited autophagy by interacting with Ulk1 and Beclin1 in the cytoplasm. Six mutants of PUMA were constructed to further study which part is responsible for the interaction, and the BH3 domain shows indispensability. When PUMA moved to mitochondria and formed a complex with Ulk1 and Bcl-X_L, which played opposite roles, in promoting mitophagy. During this process, Ser96 of PUMA was indispensable for activating mitophagy. Besides, over-expressed PUMA or Bcl-X_L promotes obvious mitophagy, and the real-time detection of lysosome and mitochondria shows fusion. Our results identified new functions and molecular mechanisms of PUMA and Bcl-X_L in autophagy and mitophagy, which supplied theoretical bases for CRC therapy and other diseases.