Integrated proteasomal and lysosomal activity shape mTOR-regulated proteome remodeling

The crucial growth regulator mTOR is suppressed during nutrient limitation, which reduces protein synthesis and activates the ubiquitin-proteasome system (UPS) and lysosomal degradation pathways. Whereas these pathways have been extensively studied individually, their integrated dynamics, including the interplay between protein synthesis and degradation, and the coordination between lysosomal and UPS pathways, remain underexplored. Here, we couple stable isotope pulse-labeling and mass spectrometry to quantify and kinetically model proteome dynamics following mTOR inhibition in cultured human cells. Using a combination of genetics and pharmacological inhibitors, we identify proteins strictly degraded by one pathway, those that undergo multimodal degradation, and others that can flexibly access the proteasome or lysosome subject to the availability of either. Our data resource, comprised of [~]5.2 million proteomic measurements, reveals that the UPS and lysosomal pathway operate with disparate kinetics, and highlights the rapid nature of lysosomal degradation. Additionally, we observe that cells coordinate the synthesis and degradation of translation initiation and elongation factors, leading to preferential synthesis from key classes of mRNA transcripts. Taken together, this work illuminates the complex, integrated pathways influencing proteostasis when mTOR is inhibited, provides a rich resource detailing the kinetics of protein synthesis and degradation, and establishes a robust methodology for measuring proteome dynamics on a per-protein basis in the context of cellular stress.