Ben-Khoud, Y. | bioRxiv Science

bioRxiv2025

Structure of ER chaperone complex GRP170-ATP-BiP suggests a new model for substrate engagement

Molecular chaperones are essential for maintaining protein homeostasis in all living cells1. In the endoplasmic reticulum (ER), BiP and GRP170 are the sole representatives of Hsp70 and Hsp110 family and are critical for ER function. GRP170 is a relatively large and unusual chaperone that possess both nucleotide exchange and chaperoning activity2. The molecular mechanism by which these chaperones collaborate to engage substrate protein and how GRP170 couples its dual functionalities are not currently known. Here, we report the 2.7 [A] cryo-electron microscopy structure of GRP170-ATP-BiP chaperone complex purified from HEK293 cells that reveals a C-terminal curved hook domain, suggesting a role in substrate engagement in coordination with BiP. Additionally, we uncover the structural basis for GRP170 pseudo-ATPase chaperone activity - making it, to our knowledge, the first chaperone with this type of regulation. Our ER chaperone complex structure, together with prior cellular data3, suggests a new paradigm for how GRP170-BiP chaperones collaborate in ER protein quality control, broadening our understanding of how BiP/Hsp70 chaperones engage with substrates.

biochemistry↗

bioRxiv2024

OST component RPN1 is a novel regulator of IRE1 RNase activity that interacts with multiple distinct IRE1 and PERK complexes

The unfolded protein response (UPR) is an essential cell signalling system that regulates ER protein homeostasis. IRE1 and PERK are receptor proteins that propagate the UPR signal from the ER to the cytosol. Both receptors are suggested to interact with various proteins from different biological pathways, although the scale and scope of such interactions are unclear. Previous reconstitution experiments have utilized purified isolated domains of IRE1 and PERK to understand mechanism. Here, we affinity purify full length IRE1 and PERK from mammalian cells and characterise the complexes they form by biochemical techniques and assess RNase function in vivo. We identify RPN1 as a novel interacting protein present in complexes with IRE1 and PERK. In the Drosophila eye, RNAi knockdown of RPN1 results in loss of IRE1 RNase activity. This work provides a basis for understanding of protein interaction networks for IRE1 and PERK and identifies OST subunit RPN1 as a novel regulator of IRE1 RNase activity.

biochemistry↗