JCS039727 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Transport of MHC molecules via TfnR-free early endocytic carriers to TfnR-containing endosomes. Surface MHC I, MHC II or TfnR of M12.C3.F6 were labeled with monoclonal antibodies (mAbs) 30-5-7, 40F or C2 and internalized for the indicated times. Internalization was stopped by fixation with 4% paraformaldehyde at 37°C to preserve the cold-sensitive tubular structures of the CLIC/GEEC endocytic pathway. Primary antibodies were detected by cross-adsorbed secondary reagents after permeabilization. Equatorial confocal sections were selected for analysis. Note that internalized MHC molecules did not colocalize with TfnR at 5 minutes of endocytosis but showed extensive overlap after 15 minutes. Also note that endocytosed MHC II was present in tubular structures (white arrows) similar to the ones detected in the CLIC/GEEC endocytosis pathway. Tubular structures were rarely detected with conventional fixation procedures involving washes with cold HBSS before fixation. Scale bars, 2 µm.

• Supplemental Figure S2 -

  Fig. S2. Analysis of early endocytic carriers of MHC I and MHC II by density gradient electrophoresis (DGE). M12.C3.F6 cells were subjected to reversible surface biotinylation with NHSS-SS-biotin and allowed to endocytose for 5 minutes. After removal of surface biotin, the cells were processed for DGE and fractionated. From each fraction, MHC I and MHC II were retrieved by sequential immunoprecipitation. The distribution of endocytosed MHC molecules was revealed by immunoblotting against neutravidin-HRP (lower panel). (Top panels) Invariant chain is mainly distributed to the endoplasmic reticulum (ER) of M12.C3.F6 cells with only small amounts in endosomes or at the cell surface (R.L., unpublished). Its peak therefore denotes the position of the ER (fractions 13-15). β-hexosaminidase and surface TfnR represent the distribution of late endocytic organelles (lysosomes and late endosomes) and the plasma membrane, respectively. MHC II distributes to the plasma membrane and endocytic organelles (see Fig. 1, lower row; data not shown). A more detailed characterization of the DGE profile of M12.C3.F6 cells has been published previously (Lindner, 2001; Lindner, 2002). Note that briefly endocytosed MHC I and MHC II distribute to broadly overlapping zones but peak at slightly different positions.

• Supplemental Figure S3 -

  Fig. S3. Quantification of endocytosis of MHC I and MHC II. Serial dilutions of a pool of biotinylated surface protein (t=0-8 minutes, no GSH) and an undiluted sample of endocytosed protein (t=8 minutes, +GSH) were electrophoresed in the same gel and western blotted against Neutravidin-HRP. Integrated densitometry readings of suitably exposed ECL films were obtained after analysis with ImageJ. From those values, the fraction of internalized protein (t=8 minutes) relative to total protein at the cell surface was estimated using linear regression analysis. All other endocytosis time points were quantified relative to the 8-minute time point. HC, MHC I heavy chain; LC, MHC I light chain; α,β, α- and β-subunits of MHC II.

• Supplemental Figure S4 -

  Fig. S4. Quantitative comparison of invariant chain (Ii)-driven endocytosis of MHC II versus internalization of invariant chain-free mature MHC II. M12.C3.F6 cells were surface-biotinylated with NHSS-SS-biotin and allowed to endocytose for 4-16 minutes. All samples were quantitatively GSH-stripped and subjected to sequential immunoprecipitation (IP) with the mAb In-1 followed by mAb 40F. Independent experiments ascertained that both antibodies immunoprecipitate with similar high efficiency (data not shown). Note that the amount of MHC II endocytosed via invariant chain was ≤1% of the MHC II molecules endocytosed independently of invariant chain. Thus invariant chain-driven endocytosis of MHC II contributed only negligibly to the total endocytosis of this molecule in M12.C3.F6 cells. Data from two independent experiments are shown.

• Supplemental Figure S5 -

  Fig. S5. Low TX-100 resistance of MHC I and MHC II. Post-nuclear membranes of M12.C3.F6 cells were extracted in 1% Triton X (TX)-100 for 1 hour on ice and then floated in linear 10-40% sucrose gradients in the presence (right) or absence (left) of detergent. Protein distribution was assessed by enzyme assay (AP), dot blot (MHC I) or western blot (others). Note that a low percentage of MHC proteins floated to identical positions in the absence of detergent from the gradient (left), whereas no flotation of these proteins was visible in the presence of detergent (right). AP, alkaline phosphatase; Ii_mat, mature invariant chain; Ii_imm, immature, ER-specific invariant chain; pMHC II, SDS-stable peptide-MHC II complexes.

• Supplemental Figure S6 -

  Fig. S6. Presence of light and dense DRMs in different mouse B cell lines. Post-nuclear membranes from M12.C3.F6, CH27 and TA3 B cells were extracted according to the rigorous Brij 98 protocol and floated on linear 10-40% sucrose gradients containing Brij 98. Eleven fractions and the resuspended pellet were analyzed by western blotting against G_M1 (CtB), Lamp1 (mAb 1D4B), the tetraspanin CD81 (mAb EAT-1) and MHC II (mAb 10.2.16), or by dot blotting against MHC I (mAb R1-9.6). The total protein content was analyzed by silver staining after electrophoresis. Note that the position of light and dense DRMs in the sucrose gradients varied between the cell lines but in all cases correlated with the distribution of MHC II and MHC I, respectively.

• Supplemental Figure S7 -

  Fig. S7. Ultrastructure of light and dense DRMs. Negative staining of (A) light DRMs (pool fractions 5-7, see Fig. 6A) and (C) dense DRMs (pool fractions 11-13, see Fig. 6A). Ultrathin sections of pelleted light DRMs (B) and dense DRMs (D). The inset in C shows the composite structure of a large particle. The inset in B shows the unit membrane structure of light DRMs, the one in D is of an equivalent magnification to the one in B. Scale bars, 100 nm.

• Supplemental Figure S8 -

  Fig. S8. Clustering-induced density shifts of DRMs. MHC molecules of M12.C3.F6 cells were patched with mAbs R1-9.6 (X-MHC I) or 40F (X-MHC II) and secondary antibodies, or left untreated (con). PNMs of all cells were subjected to rigorous Brij 98 extraction and flotation analysis. Data from one of two independent experiments with similar results are shown. (A, top panel) Distribution of MHC, AP and G_M1. Note that ∼30% of AP and G_M1 co-shifted with patched MHC II, whereas only a small affect on these markers or on mature MHC II was evident upon MHC I patching. (A, bottom panel) Specificity of clustering-induced shifts. Note that neither B220 nor the ER-located immature MHC II α-chain was affected under any patching condition. (B) Identification of polypeptides co-shifting with clustered MHC proteins by silver staining. Arrow denotes a polypeptide co-shifting with MHC I, asterisks denote polypeptides co-shifting with MHC II.