The integrity of a cell’s proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.

细胞蛋白质组的完整性取决于伴侣蛋白对多肽的正确折叠。伴侣蛋白 TCP-1 环状复合物 (TRiC) 充当 >10% 的细胞溶质蛋白的专用文件夹，包括细胞骨架蛋白肌动蛋白和微管蛋白。尽管其结构及其识别折叠基板的方式正在从结构研究中浮现出来，但 TRiC 室内基板的后续命运尚未确定。我们在不同的折叠阶段用底物（肌动蛋白、微管蛋白）和辅伴侣 (PhLP2A) 捕获了内源性人类 TRiC，用于通过低温 EM 进行结构测定。客户蛋白的已经折叠区域锚定在室壁上，将非结构化区域定位到中央空间以实现它们的天然折叠。在折叠周期中，基板与腔室的不同部分接合，耦合到 TRiC 开闭转换。此外，辅伴侣 PhLP2A 调节折叠，充当底物和 TRiC 室之间的分子支柱。我们的结构快照将 TRiC 中客户端蛋白质折叠的新兴模型拼凑在一起。