GRAF1 is a Novel Mediator of Parkin-dependent Mitophagy

Selective autophagic removal of damaged mitochondria by Parkin-mediated mitophagy is essential for metabolic homeostasis of long-lived cells and defects in this clearance pathway leads to the accumulation of toxic intermediates and accelerates the pathogenesis of numerous cardiometabolic diseases. This process is facilitated by the recruitment of the autophagosomal protein, LC3, by LC3-binding receptors that accumulate on damaged mitochondria. However, little is known about the precise LC3-receptors that mediate Parkin-dependent mitophagy in cardiomyocytes. Having previously identified a cardioprotective role for GRAF1, our current data indicate that GRAF1 plays critical role in promoting Parkin-dependent mitochondrial clearance. GRAF1 is expressed at high levels in the heart from E17 onwards and is poised to co-regulate actin- and lipid-dynamics by virtue of its multi-domain structure that includes a lipid binding/bending BAR domain, a phospholipid binding PH domain, a Rho-GAP domain, and a protein-interaction SH3 domain. Importantly, GRAF1 depletion in primary cardiomyocytes led to impaired mitochondrial OXPHOX-mediated ATP generation, mitochondrial membrane depolarization, increased mitochondrial-associated ROS, and increased ischemia/reperfusion-dependent myocyte death. These outcomes were accompanied by reduced LC3 mediated autophagic flux and accumulation of damaged mitochondria (and Parkin) in GRAF1 deficient cardiomyocytes and hearts. Mechanistically, we showed that GRAF1 facilitates Parkin-dependent mitophagy by serving as a novel LC3 receptor. GRAF1 is recruited to damaged mitochondria, is phosphorylated in a PINK1/Parkin-dependent fashion, and physically interacts with Parkin and LC3. While multiple LC3 receptors are recruited to depolarized mitochondria and act in a redundant function to promote mitochondrial engulfment, our studies reveal a unique and indispensable role for GRAF1 in this process. Our studies reveal that GRAF1 is distinctive because it facilitates mitochondrial clearance by further facilitating recruitment of Parkin and additional down-stream receptors to damaged mitochondria in a feed-forward fashion.