Protein quality control depends on the tight regulation of interactions between molecular chaperones and polypeptide substrates. Substrate release from the chaperone Hsp70 is triggered by nucleotide-exchange factors (NEFs) that control folding and degradation fates via poorly understood mechanisms. We found that the armadillo-type NEFs budding yeast Fes1 and its human homolog HspBP1 employ flexible N-terminal release domains (RDs) with substrate-mimicking properties to ensure the efficient release of persistent substrates from Hsp70. The RD contacts the substrate-binding domain of the chaperone, competes with peptide substrate for binding and is essential for proper function in yeast and mammalian cells. Thus, the armadillo domain engages Hsp70 to trigger nucleotide exchange, whereas the RD safeguards the release of substrates. Our findings provide fundamental mechanistic insight into the functional specialization of Hsp70 NEFs and have implications for the understanding of proteostasis-related disorders, including Marinesco–Sjögren syndrome.

蛋白质质量控​​制取决于分子伴侣和多肽底物之间相互作用的严格调节。分子伴侣Hsp70的底物释放是由核苷酸交换因子（NEF）触发的，该因子通过人们不了解的机制来控制折叠和降解命运。我们发现，犰狳型NEFs发芽酵母Fes1及其人类同源物HspBP1采用具有底物模拟特性的柔性N末端释放域（RDs），以确保从Hsp70有效地释放持久性底物。RD接触伴侣的底物结合结构域，与肽底物竞争结合，并且对于在酵母和哺乳动物细胞中的正常功能是必不可少的。因此，犰狳结构域与Hsp70结合以触发核苷酸交换，而RD保护底物的释放。