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. 2021 Jun 17;10:e68380. doi: 10.7554/eLife.68380

Figure 1. ER retrieval signal abundance and affinity are not correlated.

(a) Sequence logos for ER resident proteins with C-terminal KDEL retrieval signals and variants thereof calculated using frequency or protein abundance (Itzhak et al., 2017; Itzhak et al., 2016). (b) Combined cellular concentrations of ER resident proteins with canonical KDEL, RDEL, and HDEL retrieval sequences in HeLa cells and mouse brain. (c) Competition binding assays for [3H]-TAEHDEL and unlabelled TAEKDEL, TAERDEL, and TAEHDEL to the KDEL receptor. IC50 values for the competing peptides were used to calculate the apparent KDwith the Cheng-Prusoff equation (Cheng and Prusoff, 1973). (d) Endogenous KDEL receptor redistribution was measured in COS-7 cells in the absence (-ligand) or presence of K/R/H/A/DDEL (mScarlet-xDELsec). TGN46 was used as a Golgi marker. Scale bar is 10 µm. (e) The mean difference for K/R/H/A/DDEL comparisons against the shared no ligand control are shown as Cummings estimation plots. The individual data points for the fraction of KDEL receptor fluorescence in the Golgi are plotted on the upper axes with sample sizes and p values.

Figure 1—source data 1. Source data for the ligand-induced KDELR receptor retrieval assays in Figure 1e.

Figure 1.

Figure 1—figure supplement 1. Abundance of ER resident proteins and chaperones in human cells and mouse brain.

Figure 1—figure supplement 1.

(a) The mean concentration of ER resident chaperones with the indicated ER retrieval sequence variant is plotted in the bar graph (Itzhak et al., 2017; Itzhak et al., 2016). (b) Combined cellular concentrations of ER resident proteins with canonical KDEL, RDEL, and HDEL retrieval sequences in HeLa cells and mouse brain. (c) The mean concentration of KDELR1, KDELR2, and KDELR3 in HeLa cells and mouse brain is plotted in the bar graph (Itzhak et al., 2017; Itzhak et al., 2016). (d) Cells and media collected from HeLa S3 cells expressing the xDEL variants (mScarlet-xDELsec) indicated in the figure were Western blotted for resident ER chaperones and KDELR. (e) A bar graph of xDEL secretion showing mean ± SEM (n = 3). (f). Endogenous ER chaperone secretion was measured by western blotting after challenge with different retrieval signals, and plotted as a bar graph showing mean ± SEM (n = 3).
Figure 1—figure supplement 1—source data 1. Source data for analysis of ER protein levels in Figure 1—figure supplement 1a and c.
Figure 1—figure supplement 1—source data 2. Source data for the western blots in Figure 1—figure supplement 1d showing the regions taken for the figure.
Individual blot files are provided as a ZIP archive.
Figure 1—figure supplement 1—source data 3. Source data for analysis of ER protein levels in Figure 1—figure supplement 1e and f.
Figure 1—figure supplement 2. Retrieval specificity of KDELR1 and KDELR2.

Figure 1—figure supplement 2.

(a) Distribution of human KDELR1 and (b) KDELR2 was measured in COS-7 cells in the absence (-ligand) or presence of K/R/H/A/DDEL retrieval signals (K/R/H/A/DDELsec). TGN46 was used as a Golgi marker. Scale bar is 10 µm. The fraction of KDEL receptor localised to the Golgi was measured before (no ligand) and after challenge with different retrieval signals as indicated. Golgi signal for KDEL receptor and ligand intensity are shown on the scatter plots with sample sizes. Effect sizes are shown as the mean difference for K/R/H/A/DDEL comparisons against the shared -ligand control with sample sizes and p-values.
Figure 1—figure supplement 2—source data 1. Source data for the ligand-induced KDELR receptor retrieval assays in Figure 1—figure supplement 2.