The Hsp70-Hsp90 co-chaperone Hop/Stip1 shifts the proteostatic balance from folding towards degradation

Abstract

Hop/Stip1/Sti1 is thought to be essential as a co-chaperone to facilitate substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Despite this proposed key function for protein folding and maturation, it is not essential in a number of eukaryotes and bacteria lack an ortholog. We set out to identify and to characterize its eukaryote-specific function. Human cell lines and the budding yeast with deletions of the Hop/Sti1 gene display reduced proteasome activity due to inefficient capping of the core particle with regulatory particles. Unexpectedly, knock-out cells are more proficient at preventing protein aggregation and at promoting protein refolding. Without the restraint by Hop, a more efficient folding activity of the prokaryote-like Hsp70-Hsp90 complex, which can also be demonstrated in vitro, compensates for the proteasomal defect and ensures the proteostatic equilibrium. Thus, cells may act on the level and/or activity of Hop to shift the proteostatic balance between folding and degradation.

The Hsp70-Chaperone Machines in Bacteria

“…Such a deadlock can be resolved by the Hsp90 chaperone HtpG of E. coli (Morán Luengo et al, 2018). This cooperation between Hsp70 and Hsp90 chaperones is also found in eukaryotic cells and does not require the Hsp70-Hsp90 organizing protein Hop (Bhattacharya et al, 2020).…”

Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

“…We voluntarily limited the comparison to a double mutant only, since our data indicate that roughly 90% of Hsp90β WT should be phosphorylated on both S226 and S255. The WT and AA protein have an HA tag at the N-terminal and were expressed in Hsp90β KO cells, which allowed us to eliminate interference from WT endogenous Hsp90β that would most likely form heterodimers with mutants [ 48 ]. For negative control, we used WT Hsp90 without HA tag.…”

“…HEK293T-Hsp90βKO19 cells from Didier Picard’s lab were grown in DMEM (Thermo Fischer Scientific, San Jose, CA, USA; 41966-029) supplemented with 10% ( v / v ) FBS (Pan Biotech, Aidenbach, Germany; P40-37500), 100 units/mL penicillin and 100 μg/mL streptomycin (Thermo Fischer Scientific 15140-122), at 37 °C under 5% ( v / v ) CO 2 [ 48 ].…”

H90-10 single-chain antibody recognizes Hsp90beta by immunoprecipitation and Western blotting

“…Antigen: Both wild-type HEK293T cells, which endogenously express Hsp90b, and their Hsp90b knockout counterpart (Bhattacharya et al, 2020) were grown in Dulbecco's Modified Eagle's Medium supplemented with GlutaMAX, 10% fetal bovine serum, and penicillin/streptomycin (100 U/ml).…”