Functional Translatome Proteomics Reveal Converging and Dose-Dependent Regulation by mTORC1 and eIF2α

Regulation of the translatome is essential during stress. However, the precise sets of translational targets regulated by the key translational stress responses –integrated stress response (ISR) and mTORC1 – remain elusive. We developed multiplexed enhanced protein dynamics (mePROD) proteomics, combining dynamic SILAC, multiplexing, and signal amplification, to enable measuring acute changes in protein synthesis. Treating cells with ISR/mTORC1-modulating stressors, we showed extensive translatome modulation and ~20% of proteins synthesized at highly reduced rates. Comparing translation-deficient sub-proteomes revealed an extensive overlap demonstrating that target specificity is achieved on protein level and not by pathway activation. Titrating inhibition of cap-dependent translation confirmed that synthesis of individual proteins is controlled by intrinsic properties responsive to global translation inhibition. This study reports a method to measure translation rates at the nascent chain level and provides insight into how the ISR and mTORC1, two key cellular pathways, regulate the translatome to guide cellular survival upon stress.