Nix is critical to two distinct phases of mitophagy, reactive oxygen species-mediated autophagy induction and Parkin-ubiquitin-p62-mediated mitochondrial priming.
Damaged mitochondria can be eliminated by autophagy, i.e. mitophagy, which is important for cellular homeostasis and cell survival. Despite the fact that a number of factors have been found to be important for mitophagy in mammalian cells, their individual roles in the process had not been clearly defined. Parkin is a ubiquitin-protein isopeptide ligase able to translocate to the mitochondria that are to be removed. We showed here in a chemical hypoxia model of mitophagy induced by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP) that Parkin translocation resulted in mitochondrial ubiquitination and p62 recruitment to the mitochondria. Small inhibitory RNA-mediated knockdown of p62 significantly diminished mitochondrial recognition by the autophagy machinery and the subsequent elimination. Thus Parkin, ubiquitin, and p62 function in preparing mitochondria for mitophagy, here referred to as mitochondrial priming. However, these molecules were not required for the induction of autophagy machinery. Neither Parkin nor p62 seemed to affect autophagy induction by CCCP. Instead, we found that Nix was required for the autophagy induction. Nix promoted CCCP-induced mitochondrial depolarization and reactive oxygen species generation, which inhibited mTOR signaling and activated autophagy. Nix also contributed to mitochondrial priming by controlling the mitochondrial translocation of Parkin, although reactive oxygen species generation was not involved in this step. Deletion of the C-terminal membrane targeting sequence but not mutations in the BH3 domain disabled Nix for these functions. Our work thus distinguished the molecular events responsible for the different phases of mitophagy and placed Nix upstream of the events.
Ding
Wen-Xing
WX
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Ni
Hong-Min
HM
Li
Min
M
Liao
Yong
Y
Chen
Xiaoyun
X
Stolz
Donna B
DB
Dorn
Gerald W
GW
Yin
Xiao-Ming
XM
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D015687:Cell Hypoxia; D055785:Gene Knockdown Techniques; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000071456:Sequestosome-1 Protein; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
D048868:Adaptor Proteins, Signal Transducing; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; C501741:Nix protein, mouse; D017382:Reactive Oxygen Species; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D025801:Ubiquitin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein