Fig. 5: Folding of isolated mutants is independent of molecular chaperones. | Nature Communications

Fig. 5: Folding of isolated mutants is independent of molecular chaperones.

From: Distinct metabolic states of a cell guide alternate fates of mutational buffering through altered proteostasis

Fig. 5

a Heatmap representing relative quantification of transcripts encoding proteostasis components in WG350 and CSH4 in presence and absence of 350 mM NaCl. b Immunoblot (left panel) and image-based quantification (right panel) of DnaK and GroEL in CSH4 and WG350 in presence and absence of 350 mM NaCl (p-value > 0.05). Mean is plotted with standard deviation as error bars from four biological replicates. c Heatmap representing Wt GFP normalized log2 fold-change in GFP/mCherry fluorescence in deletion strains against that in BW for Wt, C1, and C6 GFP. d Bar graph representing mean of GFP/mCherry fluorescence of Wt, C1, and C6 GFP in CSH4 or WG350 with and without plasmids overexpressing either GroEL/ES (top) or DnaK/J/E (bottom) (p-value > 0.05). e Bar graph representing GFP/mCherry fluorescence of Wt, C1, and C6 GFP in CSH4 and WG350 in absence or presence of external stressors (42 °C—heat shock, 80 μM Paraquat- oxidative stress, 350 mM NaCl - osmotic stress). Mean is plotted with significance calculated using 2-sided Students’ t-test (*p-value 0.017; ***p-value 0.00007). f Bar graph for mean of refolding rate for unfolded C1 and C6 GFP in presence and absence of 400 nM DnaK/800 nM DnaJ/400 nM GrpE or 400 nM GroEL/800 nM GroES. g Immunoblot for GFP showing recruitment of nascently formed C1 GFP into monomeric GFP (blue box), dimeric GFP (red box) and higher order complexes (black box). The ratio of GFP in higher order complex to the amount in monomeric state is shown in bar graph. h Histogram for GFP/mCherry fluorescence of Wt and pool of GFP mutants (Pc) in BW, molecular chaperone (∆dnaJ, ∆dnaK, ∆tig) and protease (∆lon) knockout strains. i Plot for quantification of intact GFP in Chloramphenicol-chase for Wt and C1 GFP in BW, ∆dnaK, and ∆lon strains. Mean from three biological replicates is plotted. For d, e, f, and i: Standard deviations are plotted as error bars from three biological replicates. Also see Supplementary Fig. 4 (Source data are provided as source data file Fig. 5).

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