Figure 3.

Proteotoxic stress in the cytoplasm signal to phosphorylate eIF2α via Hsp70-Bag3- HRI axis

(A) Depletion of HRI reduces phosphorylation of eIF2α in response to MG132. Cells were transfected with siHRI or siControl and further were treated by MG132 (5uM, 3 h) or left untreated. Levels of phospho-eIF2⍺ were determined in cell lysates by immunoblotting with the corresponding antibody, experiment was performed independently for six times (n = 6).

(B) Scatterplot representation of individual data points for the change in phosphorylation of eIF2α in response to MG132 upon HRI depletion (shown as means ± SEM).

(C) Depletion of Bag3 significantly reduced phosphorylation of eIF2α in the presence of MG132. Experimental conditions were as in (A) and were performed six times independently (n = 6).

(D) Scatterplot representation of individual data points for the change in phosphorylation of eIF2α in response to MG132 upon Bag3 depletion, shown as means ± SEM.

(E) Schematic layout for the pulldown experiment to check the association of HRI, eIF2α to Hsp70-Bag3 complex. Parallel validation was performed by silencing the Bag3 or disrupting the Hsp70-Bag3 complex via JG-98.

(F) HRI associates with Bag3 in the pulldown assay. Association of HRI with Bag3 was assessed by expressing 6x-His-tagged Bag3-Full length and pulled it down together with associated proteins from naive cells and cells treated with MG132 for 3 h. The pulled down fraction was immunoblotted with anti-HRI antibody.

(G) Disruption of Hsp70-Bag3 complex leads to dissociation of HRI from ubiquitinated proteins. Cells were treated with JG-98 to disrupt Hsp70-Bag3 complex or left untreated. Ubiquitinated proteins were pulled down from these cells’ extracts using ubiquilin-1 affinity column (see STAR Methods) and HRI, phospho-eIF2α levels in the pulldowns were measured by immunoblotting.

(H) Level of Bag3 was checked in pulldown samples by immunoblotting. Bag3 was not pulled down in elution upon JG98 treatment.

(I) Inhibition of autophagy by hydroxycholoquine or NH₄Cl increases HRI levels. Cells were treated with indicated concentrations of the inhibitors for indicated time periods, and levels of HRI, p62, and Bag3 were assessed by immunoblotting. Experiments were performed in biological replicates (n = 3).

(J and K) Scatterplot representation of individual data points for the change in HRI and p62 levels shown as means ± SEM.

(L and M) HRI levels are increased upon Bag3 depletion, experiments were performed independently six times (n = 6), Scatterplot representation of individual data points for the change in HRI shown as means ± SEM. All quantifications of the blots were performed using ImageJ and OD ratios for each protein compared to the reference after normalization was calculated. In case of phosphor-eIF2α, additional normalization was performed against total eIF2α. All statistical analysis was performed using Graphpad (v9), In this figure, section two-way ANOVA was used to compute level of significance and taken as (∗p < 0.0332, ∗∗p < 0.0021, ∗∗∗p < 0.0002, ∗∗∗∗p < 0.0001). See also Figure S8.