Proteome stability and dynamic protein complex profiling identified EEF1A1 as a novel translation regulator of autophagy and mitophagy
Autophagy maintains cellular homeostasis by degrading and recycling intracellular components, while the selective clearance of damaged mitochondria, known as mitophagy, ensures mitochondrial quality control. Protein complexes orchestrate these processes, yet their dynamic regulation remains incompletely understood. Here, we integrate thermal proteome profiling with co-aggregation analysis to monitor protein thermal stability and complex assembly dynamics in response to two canonical inducers: Torin 1, which activates autophagy via mTOR inhibition, and CCCP, a mitochondrial uncoupler that triggers mitophagy. This endeavor provides a global view of the dynamic variations of known autophagy- and mitophagy-associated complexes, revealing their assembly state at various stages. Notably, we identify previously uncharacterized complexes containing the eukaryotic elongation factor 1 A1 (EEF1A1) that exhibit enhanced aggregation under both treatments. Functional analyses show that EEF1A1 depletion impairs autophagosome maturation and mitophagic degradation, while pulsed-SILAC demonstrates that EEF1A1 directly regulates the synthesis of core autophagy proteins. Together, these findings map the dynamic landscape of protein complex regulation during autophagy and mitophagy and uncover EEF1A1-mediated translational control as a previously unrecognized regulatory mechanism.