Human Hsp70 paralogs display selective J-domain interactions tuning proteostasis under stress.

Hsp70 chaperones are central regulators of proteostasis, mediating folding, refolding, degradation, and aggregation-prevention. Their activity is tuned by the diverse J-domain proteins (JDPs), which both recruit client proteins and stimulate Hsp70 ATP hydrolysis via interaction with a conserved J-domain. The cytosol contains four Hsp70 paralogs-the stress-inducible HSPA1A/B and HSPA6, and the constitutively expressed HSPA8. Whether these act redundantly or carry distinct cellular functions has remained unresolved. Here, we systematically map the interactions of cytosolic Hsp70s with broad-specificity JDPs to elucidate how paralog identity shapes cellular function. We found that despite the high conservation of the JDP-Hsp70 interaction sites, the affinities of these interactions and their functionality varied greatly. HSPA8 behaves as a generalist, engaging all JDP classes with comparable affinity, consistent with its housekeeping role. By contrast, HSPA1 preferentially binds canonical Class A and B JDPs, while showing only weak binding to Class B'. Therefore, under stress, HSPA1 pairs only with Class A/B JDPs to support robust protein refolding, while freeing Class B' to suppress protein aggregation in an Hsp70-independent manner. Most unexpectedly, HSPA6, the most stress-inducible paralog, binds selectivity to Class B JDPs, losing interactions with both Class A and B'. Thus, under severe stress, HSPA6 works exclusively with Class B JDPs to ensure ATP-dependent protein repair, while freeing Class A and B' JDPs to act independently of Hsp70 to protect damaged/misfolded proteins. Our findings reveal an evolved hierarchy of paralog-specific JDP couplings that dynamically rewires the Hsp70 network from active repair to protection during stress.