Figure 5.
SteD Binds to TMEM127 and Enables Interaction between TMEM127 and mMHCII
(A) Representative confocal immunofluorescence microscopy images of Mel Juso cells expressing FLAG-TMEM127 and GFP-SteD (white; upper panel) or only FLAG-TMEM127 (lower panel). Cells were fixed and labelled for total mMHCII (L243 antibody, green), FLAG-TMEM127 (anti-FLAG, red), and DNA (DAPI). Magnified boxed area shows vesicular colocalization of the three proteins (arrowheads). Scale bar, 10 μm.
(B) Coimmunoprecipitation of FLAG-TMEM127 with GFP-SteD but not GFP-SifB. HEK293ET cells expressing FLAG-TMEM127 and GFP-SteD or GFP-SifB were lysed and proteins were immunoprecipitated with GFP-trap beads. Samples were analyzed by immunoblot using anti-GFP, anti-FLAG, and anti-tubulin (Tub) antibodies. Representative of 3 independent experiments.
(C) SteD topology and location of mutants used in (D). Positions of blocks of alanine substitutions are shown as alternating blue and white stretches. Mutants impaired for binding to TMEM127 are located in transmembrane regions (red). Mutants defective for depletion of mMHCII surface levels are shown by ∗ (Bayer-Santos et al., 2016).
(D) Coimmunoprecipitation of FLAG-TMEM127 with GFP-SteD mutants defective for depletion of mMHCII surface levels. HEK293ET cells expressing FLAG-TMEM127 and GFP-SteD alanine substitution mutants (as indicated in C) were lysed and proteins were immunoprecipitated with GFP-trap beads. Samples were analyzed by immunoblot using anti-GFP, anti-FLAG, and anti-tubulin (Tub) antibodies. Representative of 3 independent experiments.
(E) Mander’s overlap coefficient of the fraction of TMEM127 positive pixels that colocalise with mMHCII positive pixels in the absence or presence of GFP-SteD. Data are representative of three independent experiments. Each dot represents the value for one cell. Error bars show mean ± SD.∗ p < 0.05 (Student’s t test).
(F) Coimmunoprecipitation of FLAG-TMEM127 by anti-mMHCII (L243) antibody in absence or presence of GFP-SteD. Mel Juso cells stably expressing FLAG-TMEM127 or FLAG-TMEM127 and GFP-SteD were lysed and proteins were immunoprecipitated with L243 antibody. Samples were analysed by immunoblot using anti-DRα (MHCII), anti-GFP, anti-FLAG, anti-transferrin receptor (TfR), and anti-tubulin (Tub) antibodies.
(G) Quantification of intensity of FLAG-TMEM127 signal in immunoprecipitates (F) relative to immunoprecipitated MHCII (DRα), in the absence or presence of GFP-SteD. Data show means ± SD from 5 independent experiments. ∗p < 0.05 (Student’s t test).