Figure 1.
3D model of EDEM1 and sequence alignment to ER class I α1–2 mannosidase template.
A. Refined structural alignment of EDEM1 to ER class I α1–2 mannosidase. EDEM_cDis; _cSS & _rms are the consensus disorder, consensus secondary structure and model root mean deviation from an optimal alpha carbon path, in Angstroms, respectively. B. The 3D model of EDEM1. The ID region is shown in dark-red. Helices are shown in red and beta structures in yellow. N-glycans are shown in green lines. A potential Man5 substrate is shown in orange. The calcium ion and the three main aminoacids assessed to be involved in substrate binding and processing are shown in magenta.
Figure 2.
Cloning of EDEM1 mutants and their half-lifes and localization.
A. Schematic representation of EDEM1 mutants. The putative transmembrane domain is oval, the mannosidase-like domain (MLD) is, the deletion of the ID region and the point mutants are shown in red. B. EDEM1, EDEM1- ΔIDR and EDEM1-AVV were expressed in HEK 293T cells. The cells were pulse labeled with [35S]-Methionine/Cysteine for 20 min and chased for 0 min. Half of the cell lysates were immunoprecipitated with anti-HA antibodies and the other half with anti-EDEM1 antibodies and analyzed by SDS-PAGE followed by autoradiography. Arrowhead indicates EDEM1 band, horizontal line highlights the two bands of EDEM1-ΔIDR and the asterisk points out EDEM1-AVV band. C. 293T cells overexpressing the three EDEM1 proteins grown in 3,5 cm culture dishes were lysed and one tenth of the cell lysate was loaded on gel separated by SDS-PAGE and blotted with goat anti-HA or rabbit anti-EDEM1 antibodies. D. Cells expressing EDEM1 or EDEM2 recombinant constructs were used for western blot experiments, similar to Figure 2C, using rabbit α-EDEM1 or rabbit α-EDEM2 antibodies as mentioned in the figure. E. EDEM1 mutants were transfected in HEK 293T cells, pulse-labeled for 30 min and chased for 0 min. Cell lysates were immunoprecipitated with rabbit anti-EDEM1 antibodies. Samples were divided in two an subjected to EndoH digestion over night and solved by SDS-PAGE. F. To determine the rate of degradation of EDEM1 mutants, HEK 293T cells overexpressing the EDEM mutants were labeled with [35S]-Methionine/Cysteine for 20 min and chased for 0 up to 6 h. Total lysates were immunoprecipitated with anti-EDEM1 antiserum and the isolated proteins were separated by SDS-PAGE and visualized by autoradiography. G. The graph shows the average percent EDEM1 remaining after chase relative to corresponding pulse, using Image J software, mean of three independent experiments. H. A375 cells were grown on coverslips for one day, transfected with cDNA encoding for the three EDEM1 mutants and processed for immunofluorescence with polyclonal anti-EDEM1 and rabbit anti-calnexin antibodies.
Figure 3.
EDEM1 expression modulates the degradation of tyrosinase mutants.
A. Schematic representation of tyrosinase mutants; the glycans are shown in black, the transmembrane domain (T) is oval and the C-terminal cytosolic tail (C) is grey. B. HEK 293T cells were co-transfected with plasmid encoding for siRNA for EDEM 1 protein and with WT-TYR or ST-TYR. Cells were pulse-labeled for 20 min, chased for the indicated time points and immunoprecipitated with anti-tyrosinase antibodies. The immunocomplexes were separated by SDS-PAGE and visualized by autoradiography. Graphs show the relative densitometry for the mentioned experiment using ImageJ. C. Tyrosinase wild type and soluble form were co transfected with EDEM1 mutants in 293T cells. One tenth of the cellular lysate was loaded on gel, transferred on membrane and blotted using mouse anti-tyrosinase (T311), goat anti-HA antibodies and goat anti-calnexin antibodies. Calnexin was used as loading control. D. Non-glycosylated tyrosinase was used for the same experiment as in B and cell lysates were used for Western blot with mouse anti-tyrosinase, rabbit anti-EDEM1 and goat anti-calnexin antibodies. E. The graph shows the decrease in tyrosinase expression relative to control sample, average of three independent experiments.
Figure 4.
Tyrosinase mutants do not associate with EDEM1 in the absence of the ID region.
A. 293T cells were co-transfected tyrosinase mutants, pulse labeled and chased for the indicated time points and the imunocomplexes were isolated using tyrosinase T311 or HA antibodies, separated on gel and visualized by autoradiography. B. The deletion mutant of EDEM1 (EDEM1-ΔIDR) was subjected to the same type of experiment as above, using tyrosinase and EDEM1 polyclonal antibodies. Mock samples were cells transfected with tyrosinase (mock 1) or EDEM1 (mock 2). C. EDEM1-AVV was co-expressed with tyrosinase wild type and soluble mutant and subsequently used for metabolic labeling for 20 min, harvested and immunoprecipitated with tyrosinase or EDEM1 antibodies. D. NG-TYR was co-transfected with EDEM1 mutants in 293T cells which were pulse labeled for 20 min and immunoprecipitated with EDEM1 or tyrosinase antibodies. As shown wild type and triple mutant of EDEM1 coprecipitate with non-glycosylated tyrosinase, but not the truncated EDEM1 mutant(EDEM1-ΔIDR). E. EDEM1 and tyrosinase mutants were expressed in HEK293T cells, pulse labeled 20 min and immunoprecipitated with corresponding antibodies as control for the previously discussed co-precipitations.
Figure 5.
Association of EDEM1 mutants with calnexin and SEL1L.
A. HEK 293T cells overexpressing EDEM1 mutants were radioactively labeled for 30 min, chased for 0 and 15 min, harvested and lysed. Cells were immunoprecipitated with anti-EDEM1 or anti-calnexin antibodies and analyzed as in Figure 4. B. (Upper panel). Cells expressing EDEM1 mutants were pulse-labeled for 20 min and chased for 1 h. Half of the cellular lysates were used for immunoprecipitation with anti-SEL1L antibodies and the other half was used for immunoprecipitation with anti-EDEM1 antiserum and analyzed as in A. EDEM1 mutants co-precipitate with SEL1L protein. (Middle panel). Cells expressing EDEM1 mutants were immunoprecipitated with anti-EDEM1 antibodies. The immunocomplexes along with total lysates (TL) controls were separated by SDS-PAGE and Western-blotted with anti- SEL1L antibodies. (Lower panel). Total lysates from the previously described experiment were used for gel electrophoresis and Western blot with EDEM1 antibodies to test the expression of overexpressed mutants. C. A375 cells expressing EDEM1 mutants were processed for immunofluorescence, as described in Experimental procedures, with anti-SEL1L and anti-EDEM1 antibodies.