Cells possess a sophisticated machinery of molecular chaperones to maintain the quality of protein folding, but this system’s capacity often declines in ageing and disease, leading to the accumulation of toxic aggregates. The Hsp70 chaperones and a network of their regulators can execute both canonical “foldase” and powerful “disaggregase” functions. Using a real-time, organelle-targetable proteostasis probe, we recently reported that the endoplasmic reticulum (ER) version of this system—BiP and its co-chaperones—activates its disaggregation capacity upon ER stress. However, the physical principle underlying this switch is unknown. The outstanding challenge is to understand how, and by what determinants, the same core machinery operates as a foldase or a disaggregase. To dissect the biophysics of this switch, we established a toolbox combining cell-based fluorescence lifetime imaging microscopy (FLIM) to probe chaperone performance with single-molecule kinetic readouts monitoring the activity of a reconstituted system in vitro. Using this approach, we find that the functional switch is sterically governed by specific, competing co-chaperones that define the timing of nucleotide hydrolysis/exchange, thereby modulating substrate engagement and dwell time. The key regulatory factors were identified by mass spectrometry under disaggregation-promoting conditions and verified in cells and in vitro. Mechanistically, we propose a physical model that considers the kinetics of chaperoning—nucleotide-state timing and substrate dwell—showing that, when BiP/Hsp70 is recruited to unfolded clients or existing aggregates, these rates determine the outcome: folding versus disaggregation. Thus, we reveal a pivotal mechanism and kinetic principle by which the Hsp70/BiP chaperone system is dynamically repurposed to clear pre-existing protein aggregates, demonstrating how disaggregation versus folding is selected within the ER. Our work uncovers a sophisticated regulatory layer controlling ER proteostasis and offers a mechanistically grounded strategy for steering Hsp70 activity, with implications for therapies targeting proteinopathies.
Avezov et al. (Sun,) studied this question.