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Early endosomes are required for major histocompatiblity complex class II transport to peptide-loading compartments - PubMed

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Early endosomes are required for major histocompatiblity complex class II transport to peptide-loading compartments

V Brachet et al. Mol Biol Cell. 1999 Sep.

Free PMC article

Abstract

Antigen presentation to CD4(+) T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19 degrees C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor-containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most alphabetaIi complexes accumulated in tubules and vesicles devoid of gamma-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

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Figures

Figure 1
Figure 1

Incubation at 19°C reversibly blocks MHC class II molecule maturation. B4-14 cells were pulsed for 20 min with [35S]methionine and chased at 19°C for 60 min (1st through 7th lanes) or for 180 min (8th lane) and shifted to 37°C for the indicated times. Positive control cells (1st, 9th, and 10th lanes) were directly chased at 37°C for the indicated times. After cell lysis, I-Ab molecules were immunoprecipitated with the Y3P mAb. The samples were not boiled before SDS-PAGE analysis. Labeled class II molecules were not detected after the 20-min pulse (1st lane) because the Y3P mAb used for immunoprecipitation does not detect immature αβ dimmers complexed with intact Ii chain.

Figure 2
Figure 2

Immunogold localization of αβ-Ii complexes after 2 h of 19°C incubation and upon shifting to 37°C. Ultrathin cryosections of Laz cells were immunogold labeled with an anti-αβ-DR rabbit serum (PAG-10) and a rabbit Ab directed against the C-terminal domain of the Ii chain (ICC5) (PAG-15). (A) Laz cells incubated at 19°C for 2h. Intense labeling is observed for the Ii chain (PAG 15) and MHC class II (PAG 10) in the Golgi apparatus. (B and C) Cells incubated at 19°C for 2 h and then shifted to 37°C for 45 min with Ii chain (PAG 15) and MHC class II (PAG 10) are visualized in electron-lucent vesicles and tubules apposed to the Golgi apparatus (B) as well as in endosomes (C). Bar, 100 nm.

Figure 3
Figure 3

Morphology of the HRP-containing compartments. Cells that had internalized HRP-Tf for 2 h at 19°C were processed for HRP detection using DAB and H2O2 and embedded in Epon. Ultrathin sections were then analyzed by electron microscopy. (A and B) B4.14 cells. (C and D) Laz cells. DAB reaction precipitates are observed in tubulovesicular structures. Bar, 500 nm.

Figure 4
Figure 4

Early endosome inactivation has no effect on ligand internalization but reduces their degradation. A6B9 cells were processed for early endosome inactivation. Control cells were submitted to the same treatment as cross-linked cells but were incubated with non–HRP-coupled Tf before testing FcγRIIb2-mediated ligand internalization and degradation. (A) 125I-2.4G2 kinetics of internalization in both control (black circles) and cross-linked cells (black squares); (b) Degradation of 125I-DNP-BSA-anti-DNP ICs determined by measuring the percentage of 20% TCA-soluble radioactivity. Each result shows the average obtained with four independent experiments.

Figure 5
Figure 5

Early endosome inactivation has no effect on the secretory pathway. Laz-B29 (A) or B4.14 (B) cells pulsed for 20 min with [35S]methionine were processed (cross-link) or not (control) for early endosome inactivation and chased at 37°C for the indicated times. (A) TfR was immunoprecipitated and treated or not treated with endo H before analysis on SDS-PAGE. (B) After each chase period, the cell surface was biotinylated, and MHC class I molecules were immunoprecipitated. Cell surface MHC class I molecules were isolated using streptavidin–agarose. Samples were then boiled and analyzed on SDS-PAGE. Early endosome cross-linking has no effect on ER to Golgi to plasma membrane transport.

Figure 6
Figure 6

Early endosome inactivation delays MHC class II molecule maturation and transport to the cell surface. Cells were processed as for Figure 5. MHC class II molecules were immunoprecipitated with the Y3P mAb in B4.14 and T2bb cells and with the L243 mAb in Laz cells. (A) B4.14 cells; the samples were not boiled before SDS-PAGE analysis to detect the compact mature forms of class II molecules. (B) Laz cells; because the L243 mAb stronglyassociates with the class II molecules, samples were boiled before analysis to dissociate class II-L243 aggregates. No class II molecules are detected before 1 h of chase in control cells and 2 h in cross-linked cells, because L243 mAb does not detect immature αβ-Ii complexes. (C) T2bb cells; samples were boiled before analysis.

Figure 7
Figure 7

Early endosome inactivation strongly delays the degradation of αβ dimer-associated Ii chain. B4.14 cells were processed as for Figure 6. After cell lysis, class II molecules were immunoprecipitated with a rabbit anti-class II serum that has a strong affinity for immature αβ dimers. Samples were boiled before SDS-PAGE analysis. Upper panel, autoradiography after a short exposure of the gel (4 d). Lower panel, longer exposure of the same gel (2 wk).

Figure 8
Figure 8

Immunogold localization of Ii chain in cross-linked cells. Laz cells were incubated at 19°C for 2 h in the presence of Tf-HRP and processed for early endosomes inactivation as before. The cells were then chased at 37°C for 45 min to induce accumulation of MHC class II in compartments en route to the endocytic pathway. Ultrathin cryosections were immunogold labeled with the anti-Ii chain Ab ICC5 (PAG15) and with the anti-γ-adaptin mAb (PAG10; upper panel) or with the anti-46-kDa M6PR Ab (PAG10; lower panel). Upper panel, note that the Ii-positive vesicles (PAG 15) are distinct from the γ-adaptin (PAG 10)–labeled vesicles. Lower panel, the vesicles and tubulovesicular structures (inset) labeled with the anti-Ii antibody are not labeled with the anti-46-kDa M6PR antibody. Bar, 200 nm.

Figure 8
Figure 8

Immunogold localization of Ii chain in cross-linked cells. Laz cells were incubated at 19°C for 2 h in the presence of Tf-HRP and processed for early endosomes inactivation as before. The cells were then chased at 37°C for 45 min to induce accumulation of MHC class II in compartments en route to the endocytic pathway. Ultrathin cryosections were immunogold labeled with the anti-Ii chain Ab ICC5 (PAG15) and with the anti-γ-adaptin mAb (PAG10; upper panel) or with the anti-46-kDa M6PR Ab (PAG10; lower panel). Upper panel, note that the Ii-positive vesicles (PAG 15) are distinct from the γ-adaptin (PAG 10)–labeled vesicles. Lower panel, the vesicles and tubulovesicular structures (inset) labeled with the anti-Ii antibody are not labeled with the anti-46-kDa M6PR antibody. Bar, 200 nm.

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