Journal: Scientific Reports

Article Title: The WRB Subunit of the Get3 Receptor is Required for the Correct Integration of its Partner CAML into the ER

doi: 10.1038/s41598-019-48363-2

Figure Lengend Snippet: In the absence of WRB, CAML forms ER membrane-associated foci, which are rapidly eliminated. ( A ) HeLa cells were co-transfected with FLAG-CAML and either WRB-HA or an empty plasmid and then treated with MG132 or vector for 6 h. After fixation and staining with anti-FLAG and anti-Cnx antibodies, cells were imaged by confocal microscopy. Merged images of the two channels are shown; the corresponding single channel images are shown in Fig. S1A. Scale bars 10 μm; insets, x4. ( B ) Acutely expressed FLAG-CAML forms foci when expressed alone. Cells were microinjected with the FLAG-CAML plasmid together with either the WRB-HA plasmid or an empty vector, as indicated, and incubated for 2 h before being processed for immunofluorescence with anti-FLAG and anti-Cnx antibodies. Scale bars 10 μm; insets, x6. (C ) FLAG-CAML foci are rapidly cleared from microinjected cells. Cells were microinjected with the FLAG-CAML plasmid together with pEGFPN1 and incubated for 2.5 h. Cells were then either fixed immediately or treated with CHX for 2 h or 4 h before being processed for immunofluorescence with anti-FLAG antibodies. The time course of decrease in the percentage of GFP-positive cells positive also for FLAG is plotted individually for two independent experiments (number of GFP-positive cells in experiment 1 and 2, respectively: time 0, 75 and 25; 2 h, 32 and 30; 4 h, 32 and 18). Representative images for these experiments are shown in Fig. S1B. (D) FLAG-CAML is integrated into membranes also when expressed in the absence of WRB-HA. Membrane fractions of HeLa cells co-transfected with FLAG-CAML and either WRB-HA or an empty plasmid were treated with Na 2 CO 3 and subjected to floatation through alkaline sucrose gradients. The collected fractions were analyzed by immunoblotting with the antibodies indicated to the right of the blots. WRB, CAML, and Cnx accumulated at the 1.6 M–0.25 M sucrose interface (fraction 2), while the soluble protein PDI remained in the load zone. The black (left) and open (right) arrowheads indicate the endogenous and transfected HA-tagged WRB, respectively. The asterisk on the right of the PDI panels indicates a non-specific band. Original, uncropped blots are shown in Supplementary Fig. .

Article Snippet: The following primary antibodies were obtained from the indicated sources: monoclonal antibodies (mAbs) against tubulin (clone B-5-1-2) and against the FLAG epitope (clone M2), Sigma-Aldrich (St Louis, MO); anti-PDI mAbs (clone 1D3), Merck-Millipore (Burlington, MA); anti-GFP polyclonals, MBL International (Woburn, MA); affinity-purified antibodies against the coiled-coil domain of WRB were from Synaptic Systems (Göttingen, Germany); polyclonal antibodies against the SDHB subunit of mitochondrial Complex II and against human calreticulin, mouse mAbs against COX IV, ThermoFisher Scientific; anti-TOM40 mouse mAbs (clone D-2), Santa Cruz (Dallas, TX).

Techniques: Membrane, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Incubation, Immunofluorescence, Western Blot

Journal: Scientific Reports

Article Title: The WRB Subunit of the Get3 Receptor is Required for the Correct Integration of its Partner CAML into the ER

doi: 10.1038/s41598-019-48363-2

Figure Lengend Snippet: A tagged CAML construct with two N-glycosylation acceptor sites near the C-terminus reports on the topology of CAML expressed in the presence or absence of WRB-HA. ( A ) Predicted CAML topology. Top: Comparison of CAML TMs predicted with different scales (TOPCONS ). Middle: The three-TM model for CAML topology predicts that the C-terminus is in the ER lumen. The attached opsin epitope is shown in blue, and its two N-glycosylation sites are indicated (G). The site in pink, because of its proximity to the membrane is expected to remain unused. Bottom: Sequence (blue) of opsin epitope attached to the C-terminal residue of CAML. The last two residues of the predicted third TM are in green. The two N-glycosylation sites are boxed in red. ( B ) Differences in glycosylation of CAML-NGlyc expressed in the presence or absence of WRB-HA. Left: Lysates from cells expressing CAML-NGlyc either alone or together with WRB-HA were treated or not with PNGase F and analysed by SDS-PAGE/IB. Right: Cells were treated with MG132 or vector for 6 h before lysis. o, *, and # indicate non-glycosylated (0-G), mono-glycosylated (1-G) and di-glycosylated (2-G) forms of CAML-NGlyc, respectively. ( C ) All three CAML-NGlyc forms are associated with membranes. Membrane fractions were treated with Na 2 CO 3 and subjected to floatation, as in Fig. . Filled and open arrowheads indicate endogenous WRB and WRB-HA, respectively. ( D ) Cells were semi-permeabilized and treated or not with PK followed by PNGase F. One fifth of the unproteolyzed samples was loaded compared to PK-treated ones. The lower panel demonstrates that lumenal PDI is inaccessible to protease, while the cytosolic Cnx epitope is lost after PK treatment. The cartoon depicts the genesis of the a–d bands detected in the blot of panel A. The 8.5 kDa and 5.5 kDa bands c and d are compatible with the three-TM model, while the 20.5 and 12.5 kDa bands a and b have the size expected if both the second and the third TMs are translocated. Original, uncropped blots of panels B left, B right, C and D are shown in Supplementary Fig. , respectively.

Article Snippet: The following primary antibodies were obtained from the indicated sources: monoclonal antibodies (mAbs) against tubulin (clone B-5-1-2) and against the FLAG epitope (clone M2), Sigma-Aldrich (St Louis, MO); anti-PDI mAbs (clone 1D3), Merck-Millipore (Burlington, MA); anti-GFP polyclonals, MBL International (Woburn, MA); affinity-purified antibodies against the coiled-coil domain of WRB were from Synaptic Systems (Göttingen, Germany); polyclonal antibodies against the SDHB subunit of mitochondrial Complex II and against human calreticulin, mouse mAbs against COX IV, ThermoFisher Scientific; anti-TOM40 mouse mAbs (clone D-2), Santa Cruz (Dallas, TX).

Techniques: Construct, Glycoproteomics, Comparison, Membrane, Sequencing, Residue, Expressing, SDS Page, Plasmid Preparation, Lysis