Abstract
J-domain proteins (JDPs) play essential roles in assisting Hsp70 function in diverse aspects of protein biogenesis. Previous work showed that JDPs act by recruiting Hsp70 to client proteins, assisting Hsp70 in client trapping, and regulating the ATPase cycle of Hsp70. Here we report that JDPs can further enhance the conformational quality of Hsp70-bound client proteins during tail-anchored membrane protein (TA) targeting. In the guided-entry-of-tail-anchored protein (GET) pathway in yeast, newly synthesized TAs are captured and solubilized by the cytosolic Hsp70 Ssa1, which further initiates sequential TA handovers to downstream chaperones, Sgt2 and Get3, to enable their delivery to the endoplasmic reticulum. We found that two J-domain proteins (JDPs), Ydj1 and Sis1, function in parallel to support the ER targeting of TAs in vivo . Biochemical analyses showed that, while Ydj1 and Sis1 differ in their ability to assist Ssa1 in TA trapping, both JDPs enhance the transfer of Ssa1-bound TA substrate to Sgt2. The ability of the JDPs to remodel the conformation and ATPase activity of Ssa1, rather than their client binding activity, is essential for enhancing the transfer competence of Ssa1-bound TAs and for supporting TA insertion in vivo. Our results define the Hsp70 cochaperone requirement for the GET pathway and demonstrate a role of JDPs in improving the conformational quality of Hsp70-bound membrane protein clients.
Highlights
J-domain proteins (JDPs) play essential roles in Hsp70 function by assisting Hsp70 in client trapping and regulating the Hsp70 ATPase cycle
We address this question in the guided entry of tailanchored protein (GET) pathway, which delivers an essential class of tail-anchored membrane proteins (TAs) harboring hydrophobic transmembrane domains (TMDs) to the ER and is highly conserved across eukaryotic organisms
Ydj1(H34Q) was expressed at a lower level than WT Ydj1, the abundance of Ydj1(H34Q) was comparable to those of WT and mutant Sis1, suggesting that the failure of Ydj1(H34Q) to rescue the ER insertion of BirA-Bos1 was not because of insufficient protein expression levels. These results show that a functional J-domain in Ydj1 and Sis1 to regulate Hsp70 activity is required for the ability of these JDPs to support efficient TA insertion
Summary
Received for publication, January 22, 2021, and in revised form, February 22, 2021 Published, Papers in Press, March 17, 2021, https://doi.org/10.1016/j.jbc.2021.100546 Hyunju Cho , Woo Jun Shim , Yumeng Liu, and Shu-ou Shan* From the Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA
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