Figure 5.
LC-MS/MS–based identification of defective proteins trapped by TanGIBLE. (A) Flag-immunoprecipitates from MG-132–treated HeLa cell lysates were blotted with anti-polyubiquitin FK2 antibody. Identical precipitates were silver-stained in Fig. S4 A. (B and C) TanGIBLE-immunoprecipitates shown in A were directly subjected to LC-MS/MS analysis. Volcano plots of TanGIBLE-immunoprecipitates identified by deep MS analysis, highlighting enriched or lost proteins with or without MG-132 protease inhibitor treatment (B). Lanes 2 and 3 shown in A correspond to “DMSO control” and “MG-132,” respectively. Volcano plots highlighting enriched or lost proteins in the 10S TanGIBLE interactome compared with authentic TanGIBLE (C). Lanes 3 and 4 shown in A correspond to “Wildtype TanGIBLE” and “10S TanGIBLE,” respectively. (D) Representative list of TanGIBLE target proteins identified by triplicate MS analyses. P values of abundance ratio (MG-132 versus DMSO treatments) were indicated. Transmembrane proteins are colored light blue and the subunits of multiprotein complexes are colored light pink. (E) Endogenous basigin with no glycosyl modifications (indicated by red arrowheads) was predominantly co-immunoprecipitated (IP) with Flag-TanGIBLE in an MG-132–dependent manner (lane 8). Note that successfully synthesized basigin (indicated by a yellow line in the input lane 3) was not detected in identical precipitates (yellow line in the IP lane 8). Basigin co-precipitations were largely compromised in the 10S TanGIBLE and TR-TUBE precipitations (lanes 9 and 10). (F) Abundance ratio (log2) histogram of TanGIBLE/TR-TUBE immunoprecipitates. TR-TUBE–preferred targets are shown on the right and TanGIBLE-preferred proteins are shown on the left. Note that ∼2.5-fold more TUBE-associated polyubiquitinated proteins were subjected to MS analyses compared to the amount of protein co-precipitated with TanGIBLE (Fig. S4 A). We therefore adjusted the threshold (cutoff) for TanGIBLE-preferred proteins accordingly. (G) Schematic of the successful assembly of glycosylated basigin into the ER. Note that failure of ER assembly resulted in glycosylation defects, and non-glycosylated defective basigins were trapped by the TanGIBLE. Source data are available for this figure: SourceData F5.

LC-MS/MS–based identification of defective proteins trapped by TanGIBLE. (A) Flag-immunoprecipitates from MG-132–treated HeLa cell lysates were blotted with anti-polyubiquitin FK2 antibody. Identical precipitates were silver-stained in Fig. S4 A. (B and C) TanGIBLE-immunoprecipitates shown in A were directly subjected to LC-MS/MS analysis. Volcano plots of TanGIBLE-immunoprecipitates identified by deep MS analysis, highlighting enriched or lost proteins with or without MG-132 protease inhibitor treatment (B). Lanes 2 and 3 shown in A correspond to “DMSO control” and “MG-132,” respectively. Volcano plots highlighting enriched or lost proteins in the 10S TanGIBLE interactome compared with authentic TanGIBLE (C). Lanes 3 and 4 shown in A correspond to “Wildtype TanGIBLE” and “10S TanGIBLE,” respectively. (D) Representative list of TanGIBLE target proteins identified by triplicate MS analyses. P values of abundance ratio (MG-132 versus DMSO treatments) were indicated. Transmembrane proteins are colored light blue and the subunits of multiprotein complexes are colored light pink. (E) Endogenous basigin with no glycosyl modifications (indicated by red arrowheads) was predominantly co-immunoprecipitated (IP) with Flag-TanGIBLE in an MG-132–dependent manner (lane 8). Note that successfully synthesized basigin (indicated by a yellow line in the input lane 3) was not detected in identical precipitates (yellow line in the IP lane 8). Basigin co-precipitations were largely compromised in the 10S TanGIBLE and TR-TUBE precipitations (lanes 9 and 10). (F) Abundance ratio (log2) histogram of TanGIBLE/TR-TUBE immunoprecipitates. TR-TUBE–preferred targets are shown on the right and TanGIBLE-preferred proteins are shown on the left. Note that ∼2.5-fold more TUBE-associated polyubiquitinated proteins were subjected to MS analyses compared to the amount of protein co-precipitated with TanGIBLE (Fig. S4 A). We therefore adjusted the threshold (cutoff) for TanGIBLE-preferred proteins accordingly. (G) Schematic of the successful assembly of glycosylated basigin into the ER. Note that failure of ER assembly resulted in glycosylation defects, and non-glycosylated defective basigins were trapped by the TanGIBLE. Source data are available for this figure: SourceData F5.

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