Fig 2: Calnexin interacts with nonglycosylated membrane proteins in a chaperone-like manner Immunoblot analysis of overexpressed and ALFA-tagged CNX in CNX knockout cell lines clones #1 and #2, which were used for mass spectrometry. Endogenous CNX level of wildtype HEK293T cells is shown as a control.Schematic of CNX showing its lysine residues as blue dots. Lysines can be crosslinked by DSSO. The exact location of lysine residues is pictured in the crystal structure of the lumenal domain of CNX whereas the position of lysines in the cytoplasmic tail (C-tail) is an approximation.Volcano plots derived from LC–MS/MS analyses of ALFA-tagged CNX, immunoprecipitated in 1% NP-40 buffer from transfected CNX-deficient HEK293T cells after DSSO crosslinking and compared to empty vector (EV) control co-IPs. Depicted is an enlarged section of the associated complete volcano plot in the upper right. Among the significantly enriched proteins, annotations are Uniprot gene names. CNX is shown in green. ER proteins with the GO-term annotation “endoplasmic reticulum”, which are localized to the ER lumen, are highlighted in blue and among those several known CNX interaction partners are indicated with their names. Membrane proteins are shown in red and those which are nonglycosylated are additionally labeled with their gene names. Further details on the classification of the identified membrane proteins can be found in Appendix Fig S1B. Cut-off values (solid lines) in the volcano plot were defined as log2 = > 1 (2-fold enrichment) and -log10 = (P-value) > 1.3 (P < 0.05).Schematic of Cx32 structure and its orientation within the membrane. Positions of mutations analyzed in this study are indicated with stars. The overall topology and loop lengths were obtained from the UniProt server (human GJB1 gene).Co-immunoprecipitations of endogenous CNX with FLAG-tagged Cx32 WT and its mutants including quantification and statistical analysis. Constructs were expressed in HEK293T cells. One representative immunoblot is shown. Monomers and dimers of Cx32 are indicated with brackets. All samples were normalized to WT (mean ± SEM, N = 4, *P-value < 0.05, two-tailed Student's t tests).Immunofluorescence images of Cx32 and its mutants. COS-7 cells were transiently transfected with the indicated FLAG-tagged Cx32 constructs and immunofluorescence microscopy was performed using anti-PDI (green) as an ER marker. Detection of Cx32 was performed using anti FLAG antibodies and suitable labeled secondary antibodies (red). Nuclei were stained with DAPI (blue). All three channels are overlayed. Images are representative of cells from at least three different biological replicates. GJ denotes gap junctions observed at cell–cell junctions of cells transfected with WT Cx32. Scale bars correspond to 20 µm. Source data are available online for this figure.

Fig 3: 4-PBA treatment reduces PDI levels and localization in Aga2 +/- primary osteoblast culture. (A,B) Representative Western blot and quantification of relative PDI levels in primary calvarial osteoblasts cultured and treated with 5nM 4-PBA for 7 days. n = 7 per treatment group. A two-way ANOVA was performed, *p < 0.05 was considered statistically significant. (C–F) Immunofluorescent images of primary osteoblasts probed with a conjugated antibody against PDI. Green = PDI, blue = DAPI, n = 6.

Fig 4: The transmembrane domain of Calnexin binds clients in the membrane Schematic of the reporter construct, where an antibody CL domain was fused to sequences of interest, to assess CNX interactions with individual Cx32 TMD segments. The four WT sequences of the Cx32-TMDs are shown. The Arg residue exchanged against Cys for BMH crosslinking is highlighted in TMD1. Predicted membrane integration energies are given using DGPred (Hessa et al, 2007). Sequences of TMD1 and TMD3 were inverted in the respective constructs to have the same amino acid composition and the same amino acids exposed to the lipid bilayer as in the corresponding Cx32 TMDs.Co-immunoprecipitations of endogenous CNX with the different CL-TMD constructs (NVT glycosylation site mutated to QVT) including quantification and statistical analysis. One representative immunoblot is shown. The asterisk (*) denotes a fraction of cleaved species as observed previously for similar constructs (Behnke et al, 2016). All samples were normalized to the construct containing TMD1 (mean ± SEM, N = 3, *P-value < 0.05, two-tailed Student's t tests).Interaction analysis of Calnexin with the first transmembrane domain of Cx32. Intracellular BMH crosslinking of transiently transfected V5-tagged CNX with the transiently transfected R26C mutant of Cx32-TMD1. In the AA mutant of CNX, the two cysteines within the CNX TMD were replaced by alanines. The species at approx. 250 kDa are possibly CNX dimers.Schematic of the minimal CNX construct (minCNX tmd ). This consists of a preprolactin (PPL) ER import sequence, followed by an N-terminal MYC tag, the monomeric red fluorescent protein mScarlet-I, the TM segment of CNX including an endogenous double lysine motif downstream of the TM region and a C-terminal FLAG-tag. Flexible linker regions connect the individual components. Parts derived from CNX are shown in orange. A control construct, minCNXLECTIN contains the entire lumenal lectin domain of CNX whereas another control, ER-Scarlet, is lacking any CNX segments. A C-terminal KDEL sequence was included in both control constructs for ER retention.COS-7 cells were transiently transfected with the indicated constructs and immunofluorescence microscopy was performed using anti-PDI (green) as an ER marker. Detection of minCNX tmd and both control constructs was carried out by mScarlet-I fluorescence (red). Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates. Scale bars correspond to 20 µm.Interaction of Cx32-TMD1 with the minCNX system. MinCNX tmd constructs and controls (see (D)) were co-transfected with Cx32-TMD1 into HEK293T cells. Interaction between the proteins was analyzed by co-immunoprecipitation experiments followed by immunoblotting. Source data are available online for this figure.