Fig. 1: LGMDD1 G/F domain mutants show variability in substrate processing. | Nature Communications

Fig. 1: LGMDD1 G/F domain mutants show variability in substrate processing.

From: Disease-associated mutations within the yeast DNAJB6 homolog Sis1 slow conformer-specific substrate processing and can be corrected by the modulation of nucleotide exchange factors

Fig. 1

a Kinetics of Rnq1 fibrillation in the presence of unseeded Rnq1 only (cyan blue), Sis1-WT (black), Sis1-F106L (yellow), Sis1-N108L (red), Sis1-D110Δ (green), or Sis1-F115I (blue) measured by ThT fluorescence assay. Inset: - Time to 50% ThT fluorescence was calculated by fitting the graph using the EC50 (Y = Bottom + (Top-Bottom)/(1 + 10^((lnEC50-X)*HillSlope)) equation in Graph pad prism. RFU is Relative fluorescence unit. Values shown are mean ± SEM, n = 3 biologically independent samples. For (a; inset) each LGMDD1 mutant was compared with Sis1-WT across and ***p < 0.00019 for Sis1-F115I, **p < 0.00446 for Sis1-N108L, *p < 0.0489 for Sis1-F106L, NS = 0.25297 (non-significant) for Sis1-D110Δ values are reported for unpaired, two-sided t-test. b The morphology of amyloid fibers formed from Rnq1 at 18 °C in vitro in the presence and absence of Sis1-WT and/or LGMDD1 mutant were imaged by TEM. Experiments were performed in triplicate, representative images shown. The scale bar represents 200 nm. c Refolding activity of heat-denatured luciferase in the presence of LGMDD1 mutants. Luciferase along with Ssa1-WT and Sis1-WT/mutants was incubated at 42 °C for 10 min to heat denature luciferase. At various time points, activity was measured by a luminometer after adding substrate. Values shown are mean + SEM, n = 3 biologically independent samples. For c; each LGMDD1 mutant was compared with Sis1-WT across all time-points and ***p < 0.001, **p < 0.01, and *p < 0.05 values are reported for unpaired, two-sided t-test. Source data are provided as a Source data file.

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