Dynamic allocation of DnaK couples proteostasis demand to chaperone availability

Protein-quality-control systems are essential for cells to maintain protein homeostasis during both steady-state growth and acute stress. Yet, how protein chaperone engagement dynamically changes as proteostasis demand increases remains poorly understood. Here, we combine rapid temperature control with single-molecule tracking to follow the bacterial heat shock protein 70 (Hsp70) DnaK in live Escherichia coli. We found that most DnaK molecules actively engage with the proteome already at the optimal growth temperature, rather than form a freely diffusing reserve. Acute heat shock reallocates the DnaK pool within seconds primarily increasing the lifetime but also frequency of client engagements. Perturbing the chaperone network reveals that this redistribution reflects proteostasis demand and network capacity: loss of small heat shock proteins IbpAB drives prolonged DnaK engagement and limits recovery, whereas overexpression of thermolabile proteins alone can produce heat shock-like dynamics. Together, our findings reveal how chaperones are dynamically reallocated during proteotoxic stress and establish DnaK mobility as a sensitive readout of proteostasis demand.