Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions
Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions
复制标题
DOI：
10.1042/bj20070084
复制
发表时间：
2007-05-15
影响因子：
4.1
通讯作者：
Johnson, Jill L.
中科院分区：
文献类型：
--
作者：
Flom, Gary;Behal, Robert H.;Johnson, Jill L.
分子伴侣Hsp（热休克蛋白）90对不同细胞客户蛋白的活性至关重要。在目前的模型中，客户蛋白从热休克蛋白70转移到热休克蛋白90的过程中，介导的辅伴侣Sti 1/Hop，这可能同时与热休克蛋白70和热休克蛋白90通过单独的TPR（tetratricopeptide repeat）域相互作用，但机制和在体内的重要性，这一功能还不清楚。在本研究中，我们使用截短形式的Sti 1来确定热休克蛋白70和热休克蛋白90相互作用所需的最小区域，以及Sti I二聚化。我们发现，TPR 1和TPR 2B都有助于Hsp 70在体内的相互作用，TPR 1和TPR 2B的突变需要破坏Sti 1与Hsp 70 Ssa 1的C-末端的体外相互作用。TPR 2A结构域是体内Hsp 90相互作用所必需的，但分离的TPR 2A结构域不足以与Hsp 90相互作用，除非与TPR 2B结构域组合。然而，分离的TPR 2A是纯化的Sti 1作为二聚体在溶液中迁移的必要和足够的。DP 2结构域，这是在体内功能所必需的，是热休克蛋白70和热休克蛋白90的相互作用，以及Sti 1二聚化。作为Sti 1在体内介导Hsp 70和Hsp 90之间相互作用的作用的证据，我们鉴定了导致Hsp 90复合物中Hsp 70恢复减少的Sti 1突变体。我们还确定了两个Hsp 90突变体，表现出减少Hsp 70的相互作用，这可能有助于澄清客户端之间的两个分子伴侣转移的机制。
The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti I dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR I and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo, but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo, we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.