Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Proc Natl Acad Sci U S A. 2012 Dec 26;109(52):21208-15. doi: 10.1073/pnas.1218836109. Epub 2012 Nov 28.
The eukaryotic chaperonin, TRiC/CCT (TRiC, TCP-1 ring complex; CCT, chaperonin containing TCP-1), uses a built-in lid to mediate protein folding in an enclosed central cavity. Recent structural data suggest an effective size limit for the TRiC folding chamber of ∼70 kDa, but numerous chaperonin substrates are substantially larger. Using artificial fusion constructs with actin, an obligate chaperonin substrate, we show that TRiC can mediate folding of large proteins by segmental or domain-wise encapsulation. Single or multiple protein domains up to ∼70 kDa are stably enclosed by stabilizing the ATP-hydrolysis transition state of TRiC. Additional domains, connected by flexible linkers that pass through the central opening of the folding chamber, are excluded and remain accessible to externally added protease. Experiments with the physiological TRiC substrate hSnu114, a 109-kDa multidomain protein, suggest that TRiC has the ability to recognize domain boundaries in partially folded intermediates. In the case of hSnu114, this allows the selective encapsulation of the C-terminal ∼45-kDa domain and segments thereof, presumably reflecting a stepwise folding mechanism. The capacity of the eukaryotic chaperonin to overcome the size limitation of the folding chamber may have facilitated the explosive expansion of the multidomain proteome in eukaryotes.
真核伴侣蛋白 TRiC/CCT（TRiC，TCP-1 环复合物；CCT，包含 TCP-1 的伴侣蛋白）使用内置的盖子在封闭的中央腔中介导蛋白质折叠。最近的结构数据表明，TRiC 折叠腔的有效尺寸限制约为 70 kDa，但许多伴侣蛋白底物要大得多。使用与肌动蛋白（一种必需的伴侣蛋白底物）的人工融合构建体，我们表明 TRiC 可以通过分段或域方式封装来介导大蛋白的折叠。通过稳定 TRiC 的 ATP 水解过渡态，可以稳定地封装单个或多个最多约 70 kDa 的蛋白质结构域。通过穿过折叠腔中央开口的柔性接头连接的其他结构域被排除在外，并保持对外加蛋白酶的可及性。使用生理伴侣蛋白 TRiC 的底物 hSnu114（一种 109 kDa 的多结构域蛋白）的实验表明，TRiC 具有识别部分折叠中间物中结构域边界的能力。就 hSnu114 而言，这允许选择性地封装 C 末端约 45 kDa 的结构域及其片段，这可能反映了逐步折叠的机制。真核伴侣蛋白克服折叠腔尺寸限制的能力可能促进了真核生物中多结构域蛋白质组的爆炸式扩张。
