The ADP/ATP translocase drives mitophagy independent of nucleotide exchange

Mitochondrial homeostasis vitally depends on mitophagy, the programmed degradation of mitochondria. The roster of proteins known to participate in mitophagy remains small. We devised here a multidimensional CRISPR/Cas9 genetic screen, using multiple mitophagy reporter systems and pro-mitophagy triggers, and uncover numerous new components of Parkin-dependent mitophagy1. Unexpectedly, we identify the adenine nucleotide translocator (ANT) complex as required for mitophagy in multiple cell types. While pharmacological inhibition of ANT-mediated ADP/ATP exchange promotes mitophagy, genetic ablation of ANT paradoxically suppresses mitophagy. Importantly, ANT promotes mitophagy independently of its nucleotide translocase catalytic activity. Instead, the ANT complex is required for inhibition of the presequence translocase TIM23, leading to PINK1 stabilization, in response to bioenergetic collapse. ANT modulates TIM23 indirectly via interaction with TIM44, known to regulate peptide import through TIM232. Mice lacking ANT1 reveal blunted mitophagy and consequent profound accumulation of aberrant mitochondria. Disease-causing human mutations in ANT1 abrogate binding to TIM44 and TIM23 and inhibit mitophagy. Together, these data identify a novel and essential function for ANT as a fundamental mediator of mitophagy in health and disease.