UC San Diego

Mitochondria are double-membraned organelles essential for ATP production through oxidative phosphorylation and play a critical role in maintaining energy metabolism and regulating cell death. Damaged mitochondria are cleared through mitophagy, a selective form of autophagy. Defects in genes involved in mitophagy contribute to the development of several early-onset neurodegenerative diseases, including Parkinson’s disease and amyotrophic lateral sclerosis (ALS). To ensure the timely removal of damaged mitochondria, cells have evolved multiple overlapping mechanisms to regulate mitochondrial quality control. Our lab has recently identified one such pathway, where the AMP-activated protein kinase (AMPK) activates mitophagy through phosphorylation of the serine/threonine kinase ULK1. Upon mitochondrial stress, AMPK is activated and phosphorylates the ubiquitin E3 ligase Parkin, a rate-limiting early step in ubiquitin-dependent mitophagy. In addition to ubiquitin-dependent mitophagy, there is also a ubiquitin-independent mitophagy mechanism, in which outer mitochondrial membrane (OMM) proteins containing LC3-interacting region (LIR) motifs directly bind Atg8 family proteins to recruit the autophagosomal membrane. BCL2L13 is one of five known outer mitochondrial membrane proteins with a LIR motif known to serve as a mitophagy receptor. Studies on the budding yeast homolog of BCL2L13, Atg32, revealed that, as was reported for other mammalian selective autophagy receptor proteins, phosphorylation at a serine residue preceding the LIR motif, was shown to enhance its interaction with Atg8 proteins, promoting mitophagy2. Although BCL2L13 was discovered in 2001, whether it too was controlled by phosphorylation and what kinases may be regulating it remained unknown. In our study, we identified two phosphorylation sites on BCL2L13 induced by mitochondrial damage, Serine 261 and Serine 275, and found that these sites are regulated by AMPK and TANK-binding kinase 1 (TBK1). Notably, these phosphorylation events do not depend on another well-studied kinase involved in autophagy, ULK1, which has been shown to directly bind Bcl2L13 and its budding yeast homolog3. This discovery marks the first identification of a ubiquitin-independent mitophagy receptor being phosphorylated in an AMPK or TBK1-dependent manner — distinguishing BCL2L13 from other mitophagy receptors like NIX, BNIP3, and FUNDC1, which are all phosphorylated adjacent to their LIR motifs by ULK1. Phosphorylation of serines 261 and 275 may be crucial for BCL2L13's role in CCCP-induced mitophagy or other mitochondrial functions. Our findings suggest that BCL2L13 has a more complex and multifaceted role in cellular processes than previously understood, opening new avenues for further research.