Mitochondrial protein import stress regulates the LC3 lipidation step of mitophagy through NLRX1 and RRBP1.

Protein import into mitochondria is a highly regulated process, yet how cells clear mitochondria undergoing dysfunctional protein import remains poorly characterized. Here we showed that mitochondrial protein import stress (MPIS) triggers localized LC3 lipidation. This arm of the mitophagy pathway occurs through the Nod-like receptor (NLR) protein NLRX1 while, surprisingly, without the engagement of the canonical mitophagy protein PINK1. Mitochondrial depolarization, which itself induces MPIS, also required NLRX1 for LC3 lipidation. While normally targeted to the mitochondrial matrix, cytosol-retained NLRX1 recruited RRBP1, a ribosome-binding transmembrane protein of the endoplasmic reticulum, which relocated to the mitochondrial vicinity during MPIS, and the NLRX1/RRBP1 complex in turn controlled the recruitment and lipidation of LC3. Furthermore, NLRX1 controlled skeletal muscle mitophagy in vivo and regulated endurance capacity during exercise. Thus, localization and lipidation of LC3 at the site of mitophagosome formation is a regulated step of mitophagy controlled by NLRX1/RRBP1 in response to MPIS.