Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2011 Apr 26;108(17):6915-20. doi: 10.1073/pnas.1102828108. Epub 2011 Apr 7.
Yeast Hsp104 and its bacterial homolog, ClpB, are Clp/Hsp100 molecular chaperones and AAA+ ATPases. Hsp104 and ClpB collaborate with the Hsp70 and DnaK chaperone systems, respectively, to retrieve and reactivate stress-denatured proteins from aggregates. The action of Hsp104 and ClpB in promoting cell survival following heat stress is species-specific: Hsp104 cannot function in bacteria and ClpB cannot act in yeast. To determine the regions of Hsp104 and ClpB necessary for this specificity, we tested chimeras of Hsp104 and ClpB in vivo and in vitro. We show that the Hsp104 and ClpB middle domains dictate the species-specificity of Hsp104 and ClpB for cell survival at high temperature. In protein reactivation assays in vitro, chimeras containing the Hsp104 middle domain collaborate with Hsp70 and those with the ClpB middle domain function with DnaK. The region responsible for the specificity is within helix 2 and helix 3 of the middle domain. Additionally, several mutants containing amino acid substitutions in helix 2 of the ClpB middle domain are defective in protein disaggregation in collaboration with DnaK. In a bacterial two-hybrid assay, DnaK interacts with ClpB and with chimeras that have the ClpB middle domain, implying that species-specificity is due to an interaction between DnaK and the middle domain of ClpB. Our results suggest that the interaction between Hsp70/DnaK and helix 2 of the middle domain of Hsp104/ClpB determines the specificity required for protein disaggregation both in vivo and in vitro, as well as for cellular thermotolerance.
酵母 Hsp104 和其细菌同源物 ClpB 是 Clp/Hsp100 分子伴侣和 AAA+ATP 酶。Hsp104 和 ClpB 分别与 Hsp70 和 DnaK 伴侣系统协作，从聚集体中回收和重新激活应激变性的蛋白质。Hsp104 和 ClpB 在热应激后促进细胞存活的作用是具有物种特异性的：Hsp104 不能在细菌中发挥作用，ClpB 不能在酵母中发挥作用。为了确定 Hsp104 和 ClpB 中促进这种特异性的区域，我们在体内和体外测试了 Hsp104 和 ClpB 的嵌合体。我们表明，Hsp104 和 ClpB 的中间域决定了 Hsp104 和 ClpB 在高温下对细胞存活的物种特异性。在体外蛋白质重激活实验中，含有 Hsp104 中间域的嵌合体与 Hsp70 协作，而含有 ClpB 中间域的嵌合体与 DnaK 功能协作。负责特异性的区域位于中间域的螺旋 2 和螺旋 3 内。此外，几个含有 ClpB 中间域螺旋 2 中氨基酸取代的突变体在与 DnaK 协作时在蛋白质解聚中存在缺陷。在细菌双杂交测定中，DnaK 与 ClpB 以及具有 ClpB 中间域的嵌合体相互作用，这表明物种特异性是由于 DnaK 与 ClpB 的中间域之间的相互作用所致。我们的结果表明，Hsp70/DnaK 与 Hsp104/ClpB 的中间域的螺旋 2 之间的相互作用决定了在体内和体外以及细胞耐热性中蛋白质解聚所必需的特异性。
