Subcellular spatiotemporal proteomics delineates distinct phases of ER stress proteostatic response

Endoplasmic reticulum (ER) stress is linked to numerous human diseases, yet the way ER stress responses alter the stressed proteome over time remains largely unexplored. The precise coordination between organellar protein quality control and clearance pathways in maintaining proteostasis during both early and prolonged ER stress is still uncertain.

In this study, we introduce a novel spatiotemporal proteomic approach called simultaneous proteome localization and turnover analysis with time resolution (SPLAT-TR) to examine the synthesis, degradation, and localization modifications of more than 4,000 proteins during early (1-4 hours) and prolonged (16-24 hours) ER stress. As ER stress intensifies, each time point exhibits unique protein translocation and clearance regulations, alongside differential organellar proteostasis and utilization of protein degradation pathways.

The response of mitochondria to ER stress displays a bimodal pattern, beginning with early activation of respiratory protein pathways that shift towards protein quality control mechanisms in prolonged stress, paralleling a decline in cellular energetics. Conversely, in the Golgi apparatus, there is a notable increase in the clearance of collagen proteins, which paradoxically coincides with an elevated synthesis of secretory pathway components, indicating removal through secretion.

Within the ER, prolonged stress triggers a remodeling of the proteome through increased synthesis of UFMylation and ER-phagy-related proteins, coupled with the selective degradation of ER membrane and microdomain proteins. Blocking UFMylation impacts ER-phagy receptor function and exacerbates stress-induced cell death. Collectively, these findings offer a comprehensive overview of the proteostasis changes in various cell compartments under unfolded protein response conditions, underscoring the potential of SPLAT-TR to elucidate time-dependent cellular events.