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. 2015 Dec 17;4:e12621. doi: 10.7554/eLife.12621

Figure 8. Functional consequences of BiP AMPylation in vitro.

Figure 8.

(A) Bar diagram of ATP hydrolysis by BiP and BiP AMPylated to completion (BiP-AMP), as reflected in phosphate release (detected colorimetrically). Samples containing either purified BiP or BiP-AMP (both at 5 µM) were incubated with 3 mM ATP for 1 hr at 30°C and free orthophosphate generated by ATP hydrolysis was measured. Samples lacking BiP or ATP report on the assay background. Bar graph shows mean absorbance values ± SD at 612 nm (A612) of the complex between free orthophosphate and the malachite green dye after background subtraction of three repeats (n = 3). (B) Bar diagram of Vmax values for basal ATPase activities of unmodified and AMPylated BiP, derived from experiments as in “A”. Mean values ± SD are shown (n = 3). (C) Measurement of J protein-stimulated ATPase activity of unmodified and AMPylated BiP. Samples of purified BiP or BiP-AMP (both at 1.5 µM) were incubated in presence of 2 mM ATP with isolated J-domain of ERdj6 (J) at the indicated concentrations for 3 hr at 30°C and released orthophosphate was detected as in “A”. The control reactions contained 4 µM of non-functional ERdj6H422Q J-domain (carrying a mutation in the critical HPD motif; J*) instead of the wildtype J-domain. Shown is the J protein-dependent change in ATP hydrolysis rate of BiP and BiP-AMP relative to their basal ATP hydrolysis rates in absence of J protein (set to 1) of four experiments (values ± SD, n = 4). (D) Plot of concentration-dependent steady-state binding of substrate peptide by unmodified or AMPylated BiP. Fluorescence polarization (FP) of 1 µM lucifer yellow-labeled BiP substrate peptide (HTFPAVLGSC) was measured after incubation with purified BiP or BiP-AMP at the indicated concentrations for 24 hr at 30°C in presence of 1 mM ADP. Mean values of a representative experiment performed in triplicates are shown. (E) Plot of time-dependent release of fluorescently-labeled substrate peptide from unmodified or AMPylated BiP, following injection of 400-fold excess of unlabeled substrate peptide. Fluorescence polarization (FP) signal of lucifer yellow-labeled substrate peptide (1 µM) bound to BiP or BiP-AMP (both at 40 µM) in presence of 1 mM ADP (as in “D”) was measured after addition of 400-fold excess of unlabeled substrate peptide (0.4 mM) at t = 0. The initial values (after background subtraction) were set to 100% and non-linear regression analysis was performed on the first 60 min of peptide competition (inset). Mean values of a representative experiment performed in triplicates are plotted on the graph. The mean dissociation rate constants (koff) ± SD for BiP = 0.037 ± 0.006 min-1 and BiP-AMP = 0.212 ± 0.021 min-1 as well as the mean half-lives (t1/2) ± SD for BiP = 19.3 ± 2.9 min and BiP-AMP = 3.3 ± 0.3 min were calculated based on three independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.12621.019

Figure 8—source data 1. Data from three independent repeats (each performed in triplicates) of the experiment presented in Figure 8E are shown.
The insert on top of each graph shows the absolute fluorescence polarization (FP) signals in arbitrary units (A.U.) of a reference sample containing only free fluorescent substrate peptide, which were used to create normalized FP traces of samples containing BiP + peptide. The initial values (after reference signal subtraction) were set to 100%. The fit to a single phase decay curve (tabulated here) was better than to a two phase model. The fit values from the three experiments were used to calculate the average values for “koff” and the half-lives. Experiment 3 is shown in Figure 8E.
DOI: http://dx.doi.org/10.7554/eLife.12621.020
elife-12621-fig8-data1.zip (427.7KB, zip)
DOI: 10.7554/eLife.12621.020