Schumacher R J, Hansen W J, Freeman B C, Alnemri E, Litwack G, Toft D O
Department of Biochemistry and Molecular Biology, Mayo Graduate School, Rochester 55905, USA.
Biochemistry. 1996 Nov 26;35(47):14889-98. doi: 10.1021/bi961825h.
The proteins required for the repair of damaged proteins in the eukaryotic cytoplasm remain largely uncharacterized. The renaturation of thermally denatured firefly luciferase readily occurs in rabbit reticulocyte lysate by an ATP-dependent process. Earlier studies had shown that this chaperoning activity could be reconstituted, in part, using purified preparations of hsp70 and hsp90. We have extended the description of this system by clarifying the importance of hsp70 and hsp90 and have tested for additional factors that enhance renaturation. Using mutant hsp70 proteins, we have shown that hsp70 is required for luciferase renaturation. We have also found that hsp70 and hsp90 preparations purified by common procedures were contaminated with low levels of DnaJ proteins that are essential for the renaturing activity. When hsp70 and hsp90 preparations free of DnaJ proteins are used, the system must be supplemented with a DnaJ protein to obtain renaturation activity. The yeast DnaJ protein, YDJ-1, was found to be very effective for this purpose. Although significant renaturation can occur with only hsp70 and DnaJ proteins, hsp90 also contributes to the renaturation process, both in the complex environment of reticulocyte lysate and in a purified system. However, using highly purified hsp90 and geldanamycin, a specific inhibitor of hsp90 function, we have determined that hsp90 is not an essential component of the renaturation system. The contribution of hsp90 to renaturation is only partially blocked by geldanamycin, suggesting that this protein may influence activity in more than one way. This study indicates that hsp70, hsp90, and DnaJ proteins function cooperatively to renature damaged proteins in the eukaryotic cytoplasm and provides a framework by which additional components can be identified and individual chaperone contributions can be investigated.
真核细胞质中修复受损蛋白质所需的蛋白质在很大程度上仍未被鉴定。热变性的萤火虫荧光素酶在兔网织红细胞裂解物中通过依赖ATP的过程很容易复性。早期研究表明，这种伴侣活性部分可以用纯化的hsp70和hsp90制剂重建。我们通过阐明hsp70和hsp90的重要性扩展了对该系统的描述，并测试了其他增强复性的因素。使用突变的hsp70蛋白，我们表明hsp70是荧光素酶复性所必需的。我们还发现，通过常规方法纯化的hsp70和hsp90制剂被低水平的对复性活性至关重要的DnaJ蛋白污染。当使用不含DnaJ蛋白的hsp70和hsp90制剂时，该系统必须补充DnaJ蛋白以获得复性活性。发现酵母DnaJ蛋白YDJ-1对此非常有效。尽管仅用hsp70和DnaJ蛋白就能发生显著的复性，但hsp90在网织红细胞裂解物的复杂环境和纯化系统中也有助于复性过程。然而，使用高度纯化的hsp90和hsp90功能的特异性抑制剂格尔德霉素，我们确定hsp90不是复性系统的必需成分。格尔德霉素仅部分阻断hsp90对复性的贡献，表明该蛋白可能以多种方式影响活性。这项研究表明，hsp70、hsp90和DnaJ蛋白协同作用使真核细胞质中的受损蛋白复性，并提供了一个框架，通过该框架可以识别其他成分并研究单个伴侣的贡献。
