Dynamic stability of Sgt2 enables selective and privileged client handover in a chaperone triad
-
1.
California Institute of Technology
Abstract
AbstractMembrane protein biogenesis poses acute challenges to protein homeostasis, and how they are selectively escorted to the target membrane is not well understood. Here we address this question in the guided-entry-of-tail-anchored protein (GET) pathway, in which tail-anchored membrane proteins (TAs) are relayed through an Hsp70-Sgt2-Get3 chaperone triad for targeting to the endoplasmic reticulum. We show that the Hsp70 ATPase cycle and TA substrate drive dimeric Sgt2 from a wide-open conformation to a closed state, in which TAs are protected by both substrate binding domains of Sgt2. Get3 is privileged to receive TA from closed Sgt2, whereas off-pathway chaperones remove TAs from open Sgt2. Sgt2 closing is less favorable with suboptimal GET substrates, which are rejected during or after the Hsp70-to-Sgt2 handover. Our results demonstrate how fine-tuned conformational dynamics in Sgt2 enable hydrophobic TAs to be effectively funneled onto their dedicated targeting factor while also providing a mechanism for substrate selection.
Copyright and License
© The Author(s) 2024. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Acknowledgement
We thank members of the Shan laboratory for comments on the manuscript and A. Siegel for assistance with the quantification of Bpa crosslinking data. This work was supported by National Institutes of Health grants R35 GM136321 to S.-o. Shan and by Dean Willard Chair funds to S.W.
Contributions
H.C., Y.L., S.S. designed research; H.C., Y.L. and S. Chandrasekar performed biochemical experiments and analyzed data; Y.L. and S. Chung performed µs-ALEX experiments and analyzed data; S.W. provided guidance for µs-ALEX analysis; H.C., Y.L. and S.S. wrote the manuscript with input from S. Chandrasekar, S. Chung, and S.W.
Data Availability
All data generated in this study are provided in the manuscript’s main text, Supplemental information, Supplementary Data 1, and Source Data file. A previously published structure of E.coli DnaK used to design fluorescent dye labeling positions is available under PDB code 4EZO. Source data are provided with this paper.
Code Availability
All mpH2MM analyses in this study were performed using the Python packages, burstH2MM (Available at https://github.com/harripd/burstH2MM) and H2MM_C (Available at https://github.com/harripd/H2MMpythonlib).
Conflict of Interest
The authors declare no competing interests.
Files
| Name | Size | Download all |
|---|---|---|
|
md5:ad4c07841bbbb41e143ffa4e6fb003d2
|
3.0 MB | Preview Download |
|
md5:0597af1ccac8f1f32cdcfb695d0d1d44
|
3.2 MB | Preview Download |
|
md5:f64bb5b0e8233dfc63ace578c38c41ee
|
7.8 MB | Preview Download |
|
md5:d347fa0f598f0c60c1abd6b6db8be8db
|
73.5 kB | Preview Download |
|
md5:d8e60af08f0cdf6e8242165b1ee7baf0
|
26.5 MB | Preview Download |
Additional details
- PMCID
- PMC10761869
- National Institutes of Health
- R35 GM136321
- University of California, Los Angeles