Zako Tamotsu, Sahlan Muhamad, Fujii Sayaka, Yamamoto Yohei Y, Tai Phan The, Sakai Kotaro, Maeda Mizuo, Yohda Masafumi
Bioengineering Laboratory, RIKEN Institute, Saitama, Japan.
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan; Department of Chemical Engineering, University of Indonesia, Depok, Indonesia.
J Mol Biol. 2016 Jun 5;428(11):2405-2417. doi: 10.1016/j.jmb.2016.04.006. Epub 2016 Apr 11.
Prefoldin is a molecular chaperone that captures an unfolded protein substrate and transfers it to a group II chaperonin. Previous studies have shown that the interaction sites for prefoldin are located in the helical protrusions of group II chaperonins. However, it does not exclude the possibility of the existence of other interaction sites. In this study, we constructed C-terminal truncation mutants of a group II chaperonin and examined the effects of these mutations on the chaperone's function and interaction with prefoldin. Whereas the mutants with up to 6 aa truncation from the C-terminus retained more than 90% chaperone activities for protecting citrate synthase from thermal aggregation and refolding of green fluorescent protein and isopropylmalate dehydrogenase, the truncation mutants showed decreased affinities for prefoldin. Consequently, the truncation mutants showed reduced transfer efficiency of the denatured substrate protein from prefoldin and subsequent chaperonin-dependent refolding. The results clearly show that the C-terminal region of group II chaperonins contributes to their interactions with prefoldin, the transfer of the substrate protein from prefoldin and its refolding.
预折叠蛋白是一种分子伴侣，它捕获未折叠的蛋白质底物并将其转移到Ⅱ型伴侣蛋白上。先前的研究表明，预折叠蛋白的相互作用位点位于Ⅱ型伴侣蛋白的螺旋突出部。然而，这并不排除存在其他相互作用位点的可能性。在本研究中，我们构建了Ⅱ型伴侣蛋白的C端截短突变体，并研究了这些突变对伴侣蛋白功能以及与预折叠蛋白相互作用的影响。从C端截短多达6个氨基酸的突变体，在保护柠檬酸合酶免受热聚集以及绿色荧光蛋白和异丙基苹果酸脱氢酶复性方面，保留了超过90%的伴侣蛋白活性，然而，截短突变体对预折叠蛋白的亲和力降低。因此，截短突变体显示出变性底物蛋白从预折叠蛋白的转移效率降低，以及随后依赖伴侣蛋白的复性效率降低。结果清楚地表明，Ⅱ型伴侣蛋白的C端区域有助于其与预折叠蛋白的相互作用、底物蛋白从预折叠蛋白的转移及其复性。
