Abstract
<jats:p>The turnover of mitochondria through mitophagy is essential for maintaining mitochondrial function and matching mitochondrial content to cellular demand. In many cells, this process is mediated by the paralogous mitochondrial receptors BNIP3 and BNIP3L, which recruit WIPI-family autophagy effectors to initiate mitophagosome formation. However, the mechanisms that activate BNIP3/L remain poorly understood. Here, we show that the kinase GSK3 directly phosphorylates BNIP3, licensing receptor activity and activating mitophagy. Phosphorylation promotes BNIP3 recruitment of WIPI proteins, thereby initiating mitophagosome formation. We identify the critical phosphorylation sites required for this regulation and demonstrate that disruption of these sites abolishes BNIP3-dependent mitophagy. Together, our findings identify phosphorylation as a molecular switch controlling BNIP3/L activity and suggest that receptor phosphorylation may temporally and spatially license mitophagosome formation and mitochondrial clearance.</jats:p>