Abstract
Hsp70 is a key cellular system counteracting protein misfolding and aggregation, associated with stress, ageing and disease. Hsp70 solubilizes aggregates and aids protein refolding through substrate binding and release cycles regulated by co-chaperones: J-domain proteins (JDPs) and Nucleotide Exchange Factors (NEFs). Here, we elucidate the collaborative impact of Hsp110 NEFs and different JDP classes throughout Hsp70-dependent aggregate processing. We show that Hsp110 plays a major role at initial stages of disaggregation, determining its final efficacy. The NEF catalyses the recruitment of thick Hsp70 assemblies onto aggregate surface, which modifies aggregates into smaller species more readily processed by chaperones. Hsp70 stimulation by Hsp110 is much stronger with Class B than Class A JDPs and requires the auxiliary interaction between Class B JDP and the Hsp70 EEVD motif. Furthermore, we demonstrate for the first time that Hsp110 disrupts the JDP-Hsp70 interaction. Such destabilisation of chaperone complexes at the aggregate surface might improve disaggregation, but also lead to the inhibition above the substoichiometric Hsp110 optimum. Thus, balanced interplay between the co-chaperones and Hsp70 is critical to unlock its disaggregating potential.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
1. We have changed the title to better reflect the major finding of the work, the importance of the NEF during the initiation of disaggregation. The new title is: Early Steps of Protein Disaggregation by Hsp70 Chaperone and Class B J-Domain Proteins are Shaped by Hsp110. 2. The subheadings were changed to include more precise information. 3. The information of protein concentrations and number of repeats has been included in figure legends. 4. The model has been changed to better reflect the findings presented in the manuscript (Figure 5). 5. We added the results of Luc-GFP reactivation (Figure 2-figure supplement 1 B) (page 7, lines 24-27). 6. Detailed information on the experimental conditions have been added to the Methods section. 7. We added controls of the BLI aggregate-binding assay with only Hsp70 and the NEF as well as with Ssa1 and Sse1 (no Sis1) and Sis1 and Sse1 (no Ssa1) (Figure 1-figure supplement 1 G) (page 5, lines 23-24). 8. We have changed the text (page 9, lines 16-22, page 13, lines 26-28) to avoid confusion regarding the effects of the Sse1-2 variant as well as the model in the Figure 5, to underline the importance of substrate release as a cause of the Hsp110-dependent inhibition. 9. We have added comments in the Discussion section on the effects of Hsp104, differences between the yeast and human Hsp70 systems and the role of Hsp110-driven substrate release in the inhibition by the co-chaperone
