Complex I Inhibition Redirects Autophagy from Bulk Turnover to Mitochondrial Clearance

Abstract

Mitochondria are both a major autophagic substrate and a principal source of signals that control autophagy, yet it is unknown whether a mitochondrial signal can specify which substrate the pathway degrades. Using metformin to inhibit Complex I in human cells, we find that autophagy is redirected rather than induced or suppressed. Inhibition shifts the NAD+/NADH ratio before perturbing the adenylate pool, separating the redox arm of the lesion from the bioenergetic arm. Single-molecule tracking of endogenous ATG13 and WIPI2 shows that initiation is preserved while WIPI2 tethering fails, generating short-lived, abortive autophagosomes; the ULK1 interactome concurrently disengages from cytoskeletal scaffolds, and Parkin-independent mitolysosomes accumulate. This response differs from mTORC1 inhibition and does not require PEN2, and AMPK is engaged non-canonically: 1/2 restrain ATG13 at initiation, while AMPK activation alone does not reproduce the WIPI2 phenotype. Occurring at therapeutic concentrations, these results identify the ULK1 complex as the step at which Complex I inhibition reassigns autophagic substrate.