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. 2023 Mar 23;14:1612. doi: 10.1038/s41467-023-37056-0

Fig. 3. Analysis of C-terminal residues and sensitivity to C-terminal epitope tagging in Hs and At ERD2.

Fig. 3

a CLSM analysis of two human ERD2 fusions (YFP-TM-HsERD2 and HsERD2-YFP constructed as described (Silva-Alvim et al., 2018) imaged in tobacco leaf epidermis cells. Notice that only the C-terminal YFP fusion causes partial ER localisation. Size marker 10 microns. b Amy-HDEL retention assays as in Fig. 2a but either comparing the two HsERD2 fluorescent variants from panel (a), or a comparison of untagged HsERD2 (WT) with the point–mutations in the HsERD2 C-terminus indicated above each lane. Notice that the C-terminal YFP fusion has completely lost biological activity. Notice also that only the LLGG mutant has lost biological activity when untagged HsERD2 is analysed. Error bars are standard deviations from 3 biological replicas. A full dose response for KKAA is provided in Supplementary Fig. 3. c C-terminal amino acid sequences of human (KDELR2) and A. thaliana ERD2B. Conserved residues are highlighted grey and the conserved di-leucine motif is highlighted bold. d CLSM analysis of selected mutants from (b) but in the YFP-TM-HsERD2 configuration. Silent mutations in panel (b) retain the Golgi localisation, while the inactive LLGG mutant displays partial ER localisation. e Schematic of C-terminal fusions to At and Hs ERD2 for functional assays (upper) and the fluorescent derivative for CLSM analysis (lower schematic). f Secretion index of Amy-HDEL, co-expressed with either wild type human or plant ERD2 compared to the three different C-terminal modifications (FLAG, c-myc, HA) in each case. Constant levels of ERD2 encoding plasmids were co-transfected (yielding 5 standard OD units). In both instances, the addition of a FLAG or c-myc tag strongly reduced function, whilst most of the activity was maintained for each ortholog after adding the HA tag. Error bars are standard deviations from 3 biological replicas. Source data are provided as a Source data file. g Localisation of human and plant ERD2 fluorescent fusions with C-terminal FLAG, c-myc and HA tags. Notice that FLAG and c-myc additions cause an ER-Golgi localisation, whilst the addition of an HA tag does not affect the Golgi localisation of YFP-TM-ERD2 for both orthologs.