The 70 kDa heat shock protein (HSP70) family of chaperones are the front line of protection from stress-induced misfolding and aggregation of polypeptides in most organisms and are responsible for promoting the stability, folding, and degradation of clients to maintain cellular protein homeostasis. Here, we demonstrate quantitative identification of HSP70 and 71 kDa heat shock cognate (HSC70) clients using a ubiquitin-mediated proximity tagging strategy and show that, despite their high degree of similarity, these enzymes have largely nonoverlapping specificities. Both proteins show a preference for association with newly synthesized polypeptides, but each responds differently to changes in the stoichiometry of proteins in obligate multi-subunit complexes. In addition, expression of an amyotrophic lateral sclerosis (ALS)-associated superoxide dismutase 1 (SOD1) mutant protein induces changes in HSP70 and HSC70 client association and aggregation toward polypeptides with predicted disorder, indicating that there are global effects from a single misfolded protein that extend to many clients within chaperone networks. Together these findings show that the ubiquitin-activated interaction trap (UBAIT) fusion system can efficiently isolate the complex interactome of HSP chaperone family proteins under normal and stress conditions.

70 kDa热休克蛋白（HSP70）分子伴侣是在大多数生物体中免受应激诱导的多肽错误折叠和聚集的保护作用的前线，并负责促进客户的稳定性，折叠和降解，以维持细胞蛋白稳态。在这里，我们证明了使用泛素介导的邻近标记策略对HSP70和71 kDa热休克同源（HSC70）客户进行定量鉴定，并显示，尽管它们具有高度相似性，但这些酶在很大程度上没有重叠的特异性。两种蛋白质均显示出与新合成的多肽缔合的偏好，但每种蛋白质对专性多亚基复合物中蛋白质化学计量的变化反应不同。此外，肌萎缩性侧索硬化症（ALS）相关的超氧化物歧化酶1（SOD1）突变蛋白的表达诱导HSP70和HSC70客户关联的改变，并朝着具有预测性疾病的多肽聚集，表明单个折叠错误的蛋白质会产生全球影响，这种影响会扩展到许多伴侣网络中的客户。这些发现共同表明，泛素激活的相互作用陷阱（UBAIT）融合系统可以在正常和压力条件下有效地分离HSP伴侣家族蛋白的复杂相互作用组。