Figure 4.
Mutagenesis of IZUMO1 and expression of mutant proteins. (A) Structure of IZUMO1 (PDB: 5B5K [Nishimura et al., 2016]) showing the four-helix bundle (in orange) containing four solvent-exposed aromatic residues (in red, F28, W88, and W113), the hinge (in light green) with the JUNO-interacting W148 (in blue), and the Ig-like domain (in teal). (B) Structure of IZUMO1 (PDB: 5B5K [Nishimura et al., 2016]) showing solvent-excluded surface colored based on molecular lipophilicity potential maps, ranging from dark cyan (hydrophilic) to dark gold (lipophilic). The aromatic residues F28, W88, and W113 are contoured in black. (C) Schematic representation of WT IZUMO1 and the mutants, maintaining the color coding for the different domains as in A. The mutants with the deletion of the transmembrane domain and cytoplasmic tail (Ecto), the deletion of the Ig-like domain (ΔIg), the point mutation W148A, and the triple mutant (FWW) are represented. (D) Representative Western blot of naive BHK cells or transfected with a plasmid encoding for WT IZUMO1, or the mutants shown in B. The different variants were detected with an anti-V5 antibody. “Surface” indicates surface biotinylation followed by affinity purification using neutravidin agarose beads; “Total” indicates the expression in whole cell extracts. Actin is used as a loading control. (E) Representative images of cells transfected with the pCI::H2B-RFP vectors encoding for WT IZUMO1 or the different mutants and subjected to an immunostaining (in white) after permeabilization with Triton X-100 using an anti-V5 antibody or without permeabilization using an antibody against the Izumo domain (Mab120). The signals for H2B-RFP and DAPI are shown in magenta and blue, respectively. The proportion of non-permeabilized cells showing surface expression was: IZUMO1WT (41.9%, n = 1,013), IZUMO1ΔIg (0%, n = 1,000), IZUMO1Ecto (0%, n = 1,000), IZUMO1W148A (60.3%, n = 1,005), IZUMO1FWW (61.7%, n = 995). Scale bars, 20 µm. Source data are available for this figure: SourceData F4.

Mutagenesis of IZUMO1 and expression of mutant proteins. (A) Structure of IZUMO1 (PDB: 5B5K [Nishimura et al., 2016]) showing the four-helix bundle (in orange) containing four solvent-exposed aromatic residues (in red, F28, W88, and W113), the hinge (in light green) with the JUNO-interacting W148 (in blue), and the Ig-like domain (in teal). (B) Structure of IZUMO1 (PDB: 5B5K [Nishimura et al., 2016]) showing solvent-excluded surface colored based on molecular lipophilicity potential maps, ranging from dark cyan (hydrophilic) to dark gold (lipophilic). The aromatic residues F28, W88, and W113 are contoured in black. (C) Schematic representation of WT IZUMO1 and the mutants, maintaining the color coding for the different domains as in A. The mutants with the deletion of the transmembrane domain and cytoplasmic tail (Ecto), the deletion of the Ig-like domain (ΔIg), the point mutation W148A, and the triple mutant (FWW) are represented. (D) Representative Western blot of naive BHK cells or transfected with a plasmid encoding for WT IZUMO1, or the mutants shown in B. The different variants were detected with an anti-V5 antibody. “Surface” indicates surface biotinylation followed by affinity purification using neutravidin agarose beads; “Total” indicates the expression in whole cell extracts. Actin is used as a loading control. (E) Representative images of cells transfected with the pCI::H2B-RFP vectors encoding for WT IZUMO1 or the different mutants and subjected to an immunostaining (in white) after permeabilization with Triton X-100 using an anti-V5 antibody or without permeabilization using an antibody against the Izumo domain (Mab120). The signals for H2B-RFP and DAPI are shown in magenta and blue, respectively. The proportion of non-permeabilized cells showing surface expression was: IZUMO1WT (41.9%, n = 1,013), IZUMO1ΔIg (0%, n = 1,000), IZUMO1Ecto (0%, n = 1,000), IZUMO1W148A (60.3%, n = 1,005), IZUMO1FWW (61.7%, n = 995). Scale bars, 20 µm. Source data are available for this figure: SourceData F4.

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