Figure 5
Analysis of the glutamate-lysine interaction. (A) In mEGFP-Tspan17, glutamate or lysine were exchanged to alanine (mutants K18A and E88A) or interchanged (K18E/E88K). Constructs are expressed in HepG2 cells, and expression levels are analysed by Western Blot analysis. Maturation of Tspan17 involves N-glycosylation and the different bands representing pre-mature (pm) and matured (m) protein are marked with an arrow (for identification of the bands see Fig. S9). (B) The actin normalized GFP signal shows a drop in expression level upon glutamate or lysine mutation (wild type values are set to 100%). The mutation with interchanged charged residues restores the expression level. The drop in Tspan17 expression and maturation is detectable shortly after expression starts (Fig. S10). (C) The maturation was calculated as ratio between mature to total protein and was compared to the wild type (set to 100%). The glutamate-/lysine-mutation leads to a drop in protein maturation, whereas the double mutation has no effect. (D) The co-localization of the mEGFP-Tspan17 constructs with the ER were analyzed by confocal microscopy, comparing the distribution of the GFP signal to an ER marker fused to RFP. For quantification, the Pearson correlation coefficient between the two channels was calculated. Exemplary images of Tspan17 wt and the mutants are shown. (E) The Pearson correlation coefficient showed an increase of ER co-localization for the glutamate-/lysine-mutants and no effect of the double mutation. Values are given as means ± SD (n = 4 for Western blot analysis and n = 3 for microscopy; for each biological replicate 10 cells were imaged). The statistical analysis was done employing a repeated measures ANOVA comparing each mutation with the wild type. The full blots are shown in the supplementary data (Fig. S20). The data analysis and illustration was performed using Fiji-ImageJ34 (https://imagej.net/) and GraphPad Prism version 6.04 for Windows (www.graphpad.com), respectively.
