Involvement of clathrin and AP-2 in the trafficking of MHC class II molecules to antigen-processing compartments

Abstract

Major histocompatibility complex class II (MHC-II) molecules are composed of two polymorphic chains, alpha and beta, which assemble with an invariant chain, Ii, in the endoplasmic reticulum. The assembled MHC-II complexes are transported to the Golgi complex and then to late endosomes/lysosomes, where Ii is degraded and alphabeta dimers bind peptides derived from exogenous antigens. Targeting of MHC-II molecules to these compartments is mediated by two dileucine-based signals in the cytoplasmic domain of Ii. These signals bind in vitro to two adaptor protein (AP) complexes, AP-1 and AP-2, which are components of clathrin coats involved in vesicle formation and cargo sorting. The physiological roles of these proteins in MHC-II molecule trafficking, however, remain to be addressed. Here, we report the use of RNA interference to examine the involvement of clathrin and four AP complexes (AP-1, AP-2, AP-3, and AP-4) in MHC-II molecule trafficking in vivo. We found that depletion of clathrin or AP-2 caused >10-fold increases in Ii expression on the cell surface and a concomitant decrease in Ii localization to endosomal/lysosomal vesicles. In addition, depletion of clathrin or AP-2 delayed the degradation of Ii and reduced the surface expression of peptide-loaded alphabeta dimers. In contrast, depletion of AP-1, AP-3, or AP-4 had little or no effect. These findings demonstrate that clathrin and AP-2 participate in MHC-II molecule trafficking in vivo. Because AP-2 is only associated with the plasma membrane, these results also indicate that a significant pool of MHC-II molecules traffic to the endosomal-lysosomal system by means of the cell surface.

Fig. 1.

Efficient knockdown of CHC and AP-μ subunits in Mel JuSo and HeLa-CIITA cells. (A) HeLa-CIITA and Mel JuSo cells were mock-treated or treated with siRNA oligonucleotides for μ1A, μ2, μ3A, and CHC. Immunoblot (IB) analysis was performed by using antibodies to the proteins indicated. (B) Immunofluorescence (IF) microscopy of mock-treated and μ4-siRNA-treated HeLa-CIITA and Mel JuSo cells with rabbit antibody to β4. (C) FACS analysis of TfR and MHC-I surface expression in HeLa-CIITA cells that were either mock-treated (open curves) or treated with siRNAs directed to the indicated proteins (shaded curves). Primary antibodies to TfR (DF 1513) and MHC-I (W6/32) were used, followed by incubation with phycoerythrin-conjugated anti-mouse IgG.

Fig. 2.

FACS analysis of Ii and MHC II in HeLa-CIITA cells depleted of CHC and AP-μ subunits. (A) FACS analysis of Ii surface expression in HeLa-CIITA cells that were either mock-treated (open curves) or treated with siRNAs to the indicated proteins (shaded curves). Staining was with primary antibody to Ii (M-B741), followed by phycoerythrin-conjugated anti-mouse IgG. (B) Results (mean ± SD) from five separate experiments were quantified and graphed as a fold increase relative to the mock experiment. (C and D) HeLa-CIITA cells were either mock-treated (open curves) or treated with siRNAs to μ2orCHC (shaded curves). Cells were stained with either the TDR31.1 antibody (to measure total MHC-II molecule surface expression) (C) or the L243 antibody (to measure peptide-loaded MHC-II molecules) (D) and analyzed by FACS.

Fig. 3.

Immunofluorescence microscopy of nonpermeabilized (A–I) and permeabilized (J–L) Mel JuSo cells that were either mock-treated or treated with siRNAs to μ2 or CHC. Cells were stained with antibodies to Ii (M-B741 or Pin-1), total MHC-II (TDR31.1), and peptide-loaded MHC-II (L243).

Fig. 4.

Effects of CHC and μ2 depletion on Ii degradation and surface expression of Ii-associated α and β chains. (A) Mock-, CHC-, or μ2-depleted Mel JuSo cells were pulse-labeled with [³⁵S]methionine-cysteine for 20 min and chased for the times indicated. Immunoprecipitations were then performed by using the Pin-1 antibody to Ii and analyzed by SDS/PAGE. (B) Mock-, CHC-, or μ2-depleted Mel JuSo cells were incubated, pulse-labeled, and chased for the indicated times in the presence of 1 mM leupeptin. Pin-1 immunoprecipitations were again performed, and the samples were resolved by SDS/PAGE. The p8–10 Ii degradation intermediates are indicated. The positions of molecular mass markers (in kDa) are indicated. (C) The results from A and B were quantified by using imagequant software (Molecular Dynamics) as a percentage of protein at time = 0. (D) HeLa-CIITA cells were either mock-treated or treated with siRNAs to μ2 or CHC as before. The cells were then surface-biotinylated, lysed, and immunoprecipitated with the Pin-1 antibody to Ii. The redissolved immunoprecipitates were then reimmunoprecipitated with the DA6.147 antibody to MHC-II. Next, the samples were analyzed by SDS/PAGE, and biotinylated proteins were detected by using strepdavidin coupled to horseradish peroxidase. The positions of molecular mass markers and the α and β chains are indicated.

Fig. 5.

HeLa-CIITA cells were either mock-treated or treated with siRNAs directed to CHC or μ2. (A) Cathepsin D activity was measured biochemically in cell lysates by using a fluorogenic substrate specific for this enzyme. Values represent the mean enzymatic activity (in arbitrary units) ± SD from three separate experiments done in triplicate. (B–D) Cathepsin K activity was visualized by fluorescence microscopy of live cells incubated with a specific fluorogenic substrate. The cells were also labeled with DAPI to stain the nuclei.