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Abstract

BNIP3 is a mitochondrial cargo receptor that specifically targets mitochondria for degradation at the autolysosome through a specialized form of autophagy called mitophagy. BNIP3 is transcriptionally upregulated in response to a number of stressors, including hypoxia, where it functions to decrease mitochondrial mass, limit ROS, and promote the efficient use of limiting metabolites and oxygen. ULK1, the catalytic component of the autophagy initiation complex, is best known for its role in stimulating general autophagy, however recent work has demonstrated that ULK1 may also promote mitophagy. The mechanism through which ULK1 specifically promotes mitophagy remains poorly understood. Here we show that ULK1 phosphorylates BNIP3 on a critical serine residue (S17) adjacent to its amino terminal LIR motif to promote its interaction with LC3 and mitophagy. Similarly, we found that ULK1 phosphorylates the BNIP3 homolog, BNIP3L, on a homologous serine residue (S35), suggestive of an expanded role of ULK1 in the specific regulation of mitophagy. Additionally, we determined that ULK1 interaction promotes the stability of BNIP3 protein by limiting its turnover at the proteasome independent of S17 phosphorylation. This stabilization of BNIP3 protein by ULK1 requires an intact BNIP3 “BH3” domain, and deletion of this domain increases BNIP3 protein levels and decreases BNIP3 turnover independent of ULK1. We found that this process of BNIP3 protein stabilization was critical to the increase in BNIP3 protein levels upon hypoxic stress, as inhibition of ULK1 blocked BNIP3 protein upregulation in response to hypoxia. Together, the work in this thesis expands our understanding of the mechanism through which ULK1 specifically regulates mitophagy through the stabilization and phosphorylation of BNIP3.

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