Extended Data Fig. 7: Specificity of PDIA6 phase separation in cells and in vitro.
From: A multichaperone condensate enhances protein folding in the endoplasmic reticulum
a, Live-cell imaging of HeLa cells transfected with a monomeric mutant25 of PDIA6–GFP. Scale bar, 20 μm. b, In vitro phase separation of Dylight 488–PDIA6 subconstructs. The EQDN mutant has been described before25 to not bind Ca2+ to the C-terminal tail (binding site III). Scale bar, 10 μm c, Residue-specific populations of the conformation P (dark blue) and O (light blue). Each bar corresponds to one methyl group in domain b. d, Combined methyl chemical shift differences between conformations P and O in domain b. The significance threshold of 2 SDs is indicated by a pink line. e, Methyl groups with substantial chemical shift difference between conformations P and O marked as pink spheres on the structure of domain b. Residues located around helix α3 show largest chemical shift differences between the two conformations. f, Electrostatic surface potential (ESP) of domain b. Helix α3 forms part of a negatively charged patch. g, SEC elution profiles (solid lines, left axis) and MALS apparent molecular mass (dotted lines, right axis) of PDIA6 WT (pink), (R142Q, K150N, R153Q) (light blue), (K150N, R153Q, K159N) (dark blue) and 5x linker mutant (grey). All proteins eluted as a dimer. h, Melting temperature of PDIA6 WT and linker mutant constructs in SEC buffer and in presence of 10 mM of Ca2+ determined by DSF. Mean and standard deviation (n = 3 technical triplicates). Colour code as in g. i, Insulin reduction by PDIA6 WT and linker mutants. Dots represent the average and the line the fit of three independent measurements (left). Insulin reduction rate of the linker mutants relative to the WT protein (right). Colour code as in g. Mean and standard deviation (n = 3 technical triplicates). j, Chaperone activity of PDIA6 WT and linker mutants assessed by the aggregation of citrate synthase as a model substrate. Colour code as in g. Mean and standard deviation (n = 3 technical triplicates). k, Quantification of condensates per cell of PDIA6 WT (same data as in Fig. 1e unstressed (us), pink) and PDIA6 5x linker mutant (grey) transfected cells (***P = 1.14·10−7, two-sided t-test), (n = 17 cells per condition pooled across three independent experiments). l, Protein expression levels of endogenous PDIA6 and transfected PDIA6–GFP in HeLa cells. Unprocessed blots are available. m, In vitro phase separation of Dylight 488–PDIA6 WT + Dylight 488–PDIA6 5x linker mutant at 1:1 stoichiometry in presence of 10 mM Ca2+ at a final protein concentration of 25 μM (left) and 50 μM (middle) and 25 μM Dylight 488–PDIA6 WT for comparison (right). Scale bar, 10 μm.
