Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
Proc Natl Acad Sci U S A. 2012 Nov 13;109(46):18833-8. doi: 10.1073/pnas.1209277109. Epub 2012 Oct 29.
The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is an ATP-fueled machine that assists protein folding. It consists of two back-to-back stacked rings formed by eight different subunits that are arranged in a fixed permutation. The different subunits of CCT are believed to possess unique substrate binding specificities that are still mostly unknown. Here, we used high-throughput microscopy analysis of yeast cells to determine changes in protein levels and localization as a result of a Glu to Asp mutation in the ATP binding site of subunits 3 (CCT3) or 6 (CCT6). The mutation in subunit CCT3 was found to induce cytoplasmic foci termed P-bodies where mRNAs, which are not translated, accumulate and can be degraded. Analysis of the changes in protein levels and structural modeling indicate that P-body formation in cells with the mutation in CCT3 is linked to the specific interaction of this subunit with Gln/Asn-rich segments that are enriched in many P-body proteins. An in vitro gel-shift analysis was used to show that the mutation in subunit CCT3 interferes with the ability of CCT to bind a Gln/Asn-rich protein aggregate. More generally, the strategy used in this work can be used to unravel the substrate specificities of other chaperone systems.
真核细胞伴侣蛋白含有 T 复合物多肽 1（CCT/TRiC）是一种由 ATP 驱动的机器，可协助蛋白质折叠。它由两个背对背堆叠的环组成，由八个不同的亚基组成，排列方式固定不变。人们认为 CCT 的不同亚基具有独特的底物结合特异性，但这些特异性在很大程度上仍然未知。在这里，我们使用高通量显微镜分析酵母细胞，以确定由于亚基 3（CCT3）或 6（CCT6）的 ATP 结合位点中的 Glu 到 Asp 突变而导致的蛋白质水平和定位的变化。发现 CCT3 亚基中的突变会诱导称为 P 体的细胞质焦点，其中未翻译的 mRNA 积累并可以降解。对蛋白质水平变化的分析和结构建模表明，具有 CCT3 突变的细胞中 P 体的形成与该亚基与富含许多 P 体蛋白的 Gln/Asn 丰富片段的特异性相互作用有关。体外凝胶迁移分析用于表明 CCT3 亚基中的突变干扰了 CCT 结合 Gln/Asn 丰富蛋白聚集体的能力。更一般地说，这项工作中使用的策略可用于揭示其他伴侣系统的底物特异性。
