Figure 2.
Detection of mislocalized TMD protein using TanGIBLE. (A and E) Schematic of the Flag-tagged IL-2Rα protein and its derivatives used in this study. The SS is indicated in green, while the TMD is indicated in red (upper panel). Kyte-Doolittle hydrophobicity plots of IL-2Rα ΔSS protein (A, lower panel). The single hydrophobicity peak at the C-terminus of IL-2Rα ΔSS corresponds exactly to the TMD (red box). The numbers on the horizontal axis denote the corresponding amino acid positions in this protein. FL: full-length. (B) Six conserved hydrophobic residues in the tail of the BUILD domain of human BAG6 (Met172, Ile173, Ile176, Leu179, Leu180, and Met183) are indicated with red characters. These are essential for the recognition of CL1 degron hydrophobicity by BAG6 Domain I (Tanaka et al., 2016). Substitution of these hydrophobic residues with hydrophilic serine (designated N200-6S fragment, indicated with blue characters) abolished its binding ability with model defective polypeptides. This mutant protein was utilized as one of the negative controls. (C) Co-immunoprecipitation (IP) of Flag-IL-2Rα ΔSS with T7-tagged TanGIBLE, N200-6S, and N200 fragments in the presence or absence of MG-132. 24 h after transfection with expression plasmids, HeLa cells were treated with 10 μM MG-132 or vehicle (DMSO) for 4 h. The precipitates formed with anti-T7 agarose were subjected to western blot analysis with anti-Flag and anti-T7 antibodies. (D) Quantification of IL-2Rα ΔSS co-immunoprecipitated with TanGIBLE in panel B. The intensity of the co-precipitated IL-2Rα ΔSS signals with the BAG6 N200 fragment was defined as the standard (as 1.0), and the relative values of IL-2Rα ΔSS co-precipitates are indicated. The data represent the mean ± SD calculated from three biologically independent experiments (n = 3). P values were calculated using Student’s t test between N200 and TanGIBLE. (F) TanGIBLE recognizes a mislocalized transmembrane protein through its unembedded TMD. A series of Flag-tagged IL-2Rα proteins were expressed in HeLa cells with T7-tagged TanGIBLE in the presence of 10 μM MG-132, and T7 immunoprecipitates were blotted with anti-Flag antibody. The smear signals (indicated with a yellow line) in the input lane represent glycosylated (and thus successfully incorporated into the ER lumen) forms of IL-2Rα. Mislocalized forms of IL-2Rα ΔSS and FL, co-immunoprecipitated with TanGIBLE, are indicated with red and yellow arrowheads, respectively. Blue arrowheads indicate mislocalized species with a deleted TMD. Source data are available for this figure: SourceData F2.

Detection of mislocalized TMD protein using TanGIBLE. (A and E) Schematic of the Flag-tagged IL-2Rα protein and its derivatives used in this study. The SS is indicated in green, while the TMD is indicated in red (upper panel). Kyte-Doolittle hydrophobicity plots of IL-2Rα ΔSS protein (A, lower panel). The single hydrophobicity peak at the C-terminus of IL-2Rα ΔSS corresponds exactly to the TMD (red box). The numbers on the horizontal axis denote the corresponding amino acid positions in this protein. FL: full-length. (B) Six conserved hydrophobic residues in the tail of the BUILD domain of human BAG6 (Met172, Ile173, Ile176, Leu179, Leu180, and Met183) are indicated with red characters. These are essential for the recognition of CL1 degron hydrophobicity by BAG6 Domain I (Tanaka et al., 2016). Substitution of these hydrophobic residues with hydrophilic serine (designated N200-6S fragment, indicated with blue characters) abolished its binding ability with model defective polypeptides. This mutant protein was utilized as one of the negative controls. (C) Co-immunoprecipitation (IP) of Flag-IL-2Rα ΔSS with T7-tagged TanGIBLE, N200-6S, and N200 fragments in the presence or absence of MG-132. 24 h after transfection with expression plasmids, HeLa cells were treated with 10 μM MG-132 or vehicle (DMSO) for 4 h. The precipitates formed with anti-T7 agarose were subjected to western blot analysis with anti-Flag and anti-T7 antibodies. (D) Quantification of IL-2Rα ΔSS co-immunoprecipitated with TanGIBLE in panel B. The intensity of the co-precipitated IL-2Rα ΔSS signals with the BAG6 N200 fragment was defined as the standard (as 1.0), and the relative values of IL-2Rα ΔSS co-precipitates are indicated. The data represent the mean ± SD calculated from three biologically independent experiments (n = 3). P values were calculated using Student’s t test between N200 and TanGIBLE. (F) TanGIBLE recognizes a mislocalized transmembrane protein through its unembedded TMD. A series of Flag-tagged IL-2Rα proteins were expressed in HeLa cells with T7-tagged TanGIBLE in the presence of 10 μM MG-132, and T7 immunoprecipitates were blotted with anti-Flag antibody. The smear signals (indicated with a yellow line) in the input lane represent glycosylated (and thus successfully incorporated into the ER lumen) forms of IL-2Rα. Mislocalized forms of IL-2Rα ΔSS and FL, co-immunoprecipitated with TanGIBLE, are indicated with red and yellow arrowheads, respectively. Blue arrowheads indicate mislocalized species with a deleted TMD. Source data are available for this figure: SourceData F2.

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