Grp94 Works Upstream of BiP in Protein Remodeling Under Heat Stress
Grp94 Works Upstream of BiP in Protein Remodeling Under Heat Stress
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DOI：
10.1016/j.jmb.2022.167762
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发表时间：
2022-08-06
影响因子：
5.6
通讯作者：
Kravats, Andrea N.
中科院分区：
文献类型：
--
作者：
Amankwah, Yaa S.;Collins, Preston;Kravats, Andrea N.
Hsp90和Hsp70是高度保守的分子伴侣，可促进通常被称为客户的底物蛋白的适当折叠和激活。这两个伴侣在功能上合作，以atp依赖的方式折叠特定的客户。在真核细胞质中，最初的客户端折叠是由Hsp70及其共同伴侣完成的，然后将客户端再折叠中间体直接转移到Hsp90进行最终的客户端处理。然而，细胞器特异性Hsp70和Hsp90协同作用的机制细节尚不清楚。本研究利用体外重折叠法研究了内质网Hsp70和Hsp90、BiP和Grp94在蛋白质重塑中的协同作用。我们发现，在较温和的变性条件下，BiP与其共同伴侣以atp依赖的方式重新折叠错误折叠的蛋白质。Grp94在这种再折叠反应中不起主要作用。然而，在更强的有利于聚集的变性条件下，Grp94以atp独立的方式结合并保持错误折叠的客户端处于折叠能力状态，以便随后被BiP系统重塑。我们还表明，Grp94和BiP的合作不仅仅是对真核细胞重折叠机制的逆转，因为Grp94和BiP的直接相互作用并不需要客户端转移。相反，ATP结合而不是Grp94的水解促进了结合的客户端的释放，然后由BiP系统拾取，随后以不依赖Grp94的方式进行再折叠。(c) 2022 Elsevier Ltd.版权所有。
Hsp90 and Hsp70 are highly conserved molecular chaperones that promote the proper folding and activation of substrate proteins that are often referred to as clients. The two chaperones functionally collaborate to fold specific clients in an ATP-dependent manner. In eukaryotic cytosol, initial client folding is done by Hsp70 and its co-chaperones, followed by a direct transfer of client refolding intermediates to Hsp90 for final client processing. However, the mechanistic details of collaboration of organelle specific Hsp70 and Hsp90 are lacking. This work investigates the collaboration of the endoplasmic reticulum (ER) Hsp70 and Hsp90, BiP and Grp94 respectively, in protein remodeling using in vitro refolding assays. We show that under milder denaturation conditions, BiP collaborates with its co-chaperones to refold misfolded proteins in an ATP-dependent manner. Grp94 does not play a major role in this refolding reaction. However, under stronger denaturation conditions that favor aggregation, Grp94 works in an ATPindependent manner to bind and hold misfolded clients in a folding competent state for subsequent remodeling by the BiP system. We also show that the collaboration of Grp94 and BiP is not simply a reversal of the eukaryotic refolding mechanism since a direct interaction of Grp94 and BiP is not required for client transfer. Instead, ATP binding but not hydrolysis by Grp94 facilitates the release of the bound client, which is then picked up by the BiP system for subsequent refolding in a Grp94-independent manner. (c) 2022 Elsevier Ltd. All rights reserved.