Limits...
Ii chain controls the transport of major histocompatibility complex class II molecules to and from lysosomes.

Brachet V, Raposo G, Amigorena S, Mellman I - J. Cell Biol. (1997)

Bottom Line: Major histocompatibility complex class II molecules are synthesized as a nonameric complex consisting of three alpha beta dimers associated with a trimer of invariant (Ii) chains.Our results suggest that alterations in the rate or efficiency of Ii chain processing can alter the postendosomal sorting of class II molecules, resulting in the increased accumulation of alpha beta dimers in lysosome-like MIIC.Thus, simple differences in Ii chain processing may account for the highly variable amounts of class II found in lysosomal compartments of different cell types or at different developmental stages.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Section de Recherche Institut National de la Santé et de la Recherche Médicale CJF-95.01, Paris, France.

ABSTRACT
Major histocompatibility complex class II molecules are synthesized as a nonameric complex consisting of three alpha beta dimers associated with a trimer of invariant (Ii) chains. After exiting the TGN, a targeting signal in the Ii chain cytoplasmic domain directs the complex to endosomes where Ii chain is proteolytically processed and removed, allowing class II molecules to bind antigenic peptides before reaching the cell surface. Ii chain dissociation and peptide binding are thought to occur in one or more postendosomal sites related either to endosomes (designated CIIV) or to lysosomes (designated MIIC). We now find that in addition to initially targeting alpha beta dimers to endosomes, Ii chain regulates the subsequent transport of class II molecules. Under normal conditions, murine A20 B cells transport all of their newly synthesized class II I-A(b) alpha beta dimers to the plasma membrane with little if any reaching lysosomal compartments. Inhibition of Ii processing by the cysteine/serine protease inhibitor leupeptin, however, blocked transport to the cell surface and caused a dramatic but selective accumulation of I-A(b) class II molecules in lysosomes. In leupeptin, I-A(b) dimers formed stable complexes with a 10-kD NH2-terminal Ii chain fragment (Ii-p10), normally a transient intermediate in Ii chain processing. Upon removal of leupeptin, Ii-p10 was degraded and released, I-A(b) dimers bound antigenic peptides, and the peptide-loaded dimers were transported slowly from lysosomes to the plasma membrane. Our results suggest that alterations in the rate or efficiency of Ii chain processing can alter the postendosomal sorting of class II molecules, resulting in the increased accumulation of alpha beta dimers in lysosome-like MIIC. Thus, simple differences in Ii chain processing may account for the highly variable amounts of class II found in lysosomal compartments of different cell types or at different developmental stages.

Show MeSH

Related in: MedlinePlus

Distribution of MHC class II, Ii chain, and lgp-B in leupeptin-treated cells. (A) Quantification of MHC class II, Ii, and lgp-B in  control and leupeptin-treated cells. The quantifications were carried out on ultrathin cryosections immunogold labeled for Ii chain  (anti–Ii cytosolic tail mAb IN1) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb  GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC  class II (rabbit anti–I-A cytoplasmic domain serum). In each case, 40 cell profiles were analyzed. (B) Immunogold localization of Ii  chain, lgp-B, and I-A in leupeptin-treated cells. (Upper panel) Ultrathin cryosections were double immunogold labeled with the Ii chain  antibody IN-1 (anti–Ii cytosolic domain; Ii CYT) and rabbit anti–I-A cytosolic tail polyclonal antibody. Ii chain accumulates in I-A–positive compartments displaying internal vesicles and electron-dense content. (Lower panel) Ultrathin cryosections were double immunogold labeled with the anti–lgp-B mAb GL2A7 and rabbit anti–I-A cytosolic tail polyclonal antibody. Class II molecules were visualized in electron-dense, lgp-B–positive compartments. The size of the gold particles is indicated. Bars, 120 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139866&req=5

Figure 7: Distribution of MHC class II, Ii chain, and lgp-B in leupeptin-treated cells. (A) Quantification of MHC class II, Ii, and lgp-B in control and leupeptin-treated cells. The quantifications were carried out on ultrathin cryosections immunogold labeled for Ii chain (anti–Ii cytosolic tail mAb IN1) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum). In each case, 40 cell profiles were analyzed. (B) Immunogold localization of Ii chain, lgp-B, and I-A in leupeptin-treated cells. (Upper panel) Ultrathin cryosections were double immunogold labeled with the Ii chain antibody IN-1 (anti–Ii cytosolic domain; Ii CYT) and rabbit anti–I-A cytosolic tail polyclonal antibody. Ii chain accumulates in I-A–positive compartments displaying internal vesicles and electron-dense content. (Lower panel) Ultrathin cryosections were double immunogold labeled with the anti–lgp-B mAb GL2A7 and rabbit anti–I-A cytosolic tail polyclonal antibody. Class II molecules were visualized in electron-dense, lgp-B–positive compartments. The size of the gold particles is indicated. Bars, 120 nm.

Mentions: We next performed double labelings for MHC class II vs the cytoplasmic portion of Ii chain (using the IN-1 mAb), as well as for class II vs lgp-B/lamp-2 (GLA27 mAb). Since both antibodies were rat monoclonal IgGs and not strongly reactive with protein A, we used a rabbit polyclonal serum directed against the cytoplasmic domain of I-A β chain to visualize MHC class II. The labeling of MHC class II observed with the polyclonal antibody was less intense than that obtained using M5.114. As established previously for isolated CIIVs (Amigorena et al., 1995), CIIVs in control cells labeled poorly with IN-1: only 23% of class II–containing compartments also stained for Ii chain cytoplasmic tail (Fig. 7 A). Similarly, the majority of these class II–containing vesicles showed no or little lgp staining under normal conditions (Fig. 7 A). In contrast, after leupeptin treatment, the number of structures containing both class II and Ii chain, or class II and lgp-B, was greatly increased, accounting for >70% of the total intracellular class II– positive structures (Fig. 7, A and B). These results confirmed that leupeptin induced the intracellular accumulation of Ii-αβ complexes and illustrated the redistribution of MHC class II molecules to lysosomes. Thus, leupeptininduced alterations in Ii chain cleavage would appear to induce class II transport to lysosomes and appearance of MIICs in cells that do not normally accumulate class II in lysosomal compartments.


Ii chain controls the transport of major histocompatibility complex class II molecules to and from lysosomes.

Brachet V, Raposo G, Amigorena S, Mellman I - J. Cell Biol. (1997)

Distribution of MHC class II, Ii chain, and lgp-B in leupeptin-treated cells. (A) Quantification of MHC class II, Ii, and lgp-B in  control and leupeptin-treated cells. The quantifications were carried out on ultrathin cryosections immunogold labeled for Ii chain  (anti–Ii cytosolic tail mAb IN1) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb  GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC  class II (rabbit anti–I-A cytoplasmic domain serum). In each case, 40 cell profiles were analyzed. (B) Immunogold localization of Ii  chain, lgp-B, and I-A in leupeptin-treated cells. (Upper panel) Ultrathin cryosections were double immunogold labeled with the Ii chain  antibody IN-1 (anti–Ii cytosolic domain; Ii CYT) and rabbit anti–I-A cytosolic tail polyclonal antibody. Ii chain accumulates in I-A–positive compartments displaying internal vesicles and electron-dense content. (Lower panel) Ultrathin cryosections were double immunogold labeled with the anti–lgp-B mAb GL2A7 and rabbit anti–I-A cytosolic tail polyclonal antibody. Class II molecules were visualized in electron-dense, lgp-B–positive compartments. The size of the gold particles is indicated. Bars, 120 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139866&req=5

Figure 7: Distribution of MHC class II, Ii chain, and lgp-B in leupeptin-treated cells. (A) Quantification of MHC class II, Ii, and lgp-B in control and leupeptin-treated cells. The quantifications were carried out on ultrathin cryosections immunogold labeled for Ii chain (anti–Ii cytosolic tail mAb IN1) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum). In each case, 40 cell profiles were analyzed. (B) Immunogold localization of Ii chain, lgp-B, and I-A in leupeptin-treated cells. (Upper panel) Ultrathin cryosections were double immunogold labeled with the Ii chain antibody IN-1 (anti–Ii cytosolic domain; Ii CYT) and rabbit anti–I-A cytosolic tail polyclonal antibody. Ii chain accumulates in I-A–positive compartments displaying internal vesicles and electron-dense content. (Lower panel) Ultrathin cryosections were double immunogold labeled with the anti–lgp-B mAb GL2A7 and rabbit anti–I-A cytosolic tail polyclonal antibody. Class II molecules were visualized in electron-dense, lgp-B–positive compartments. The size of the gold particles is indicated. Bars, 120 nm.
Mentions: We next performed double labelings for MHC class II vs the cytoplasmic portion of Ii chain (using the IN-1 mAb), as well as for class II vs lgp-B/lamp-2 (GLA27 mAb). Since both antibodies were rat monoclonal IgGs and not strongly reactive with protein A, we used a rabbit polyclonal serum directed against the cytoplasmic domain of I-A β chain to visualize MHC class II. The labeling of MHC class II observed with the polyclonal antibody was less intense than that obtained using M5.114. As established previously for isolated CIIVs (Amigorena et al., 1995), CIIVs in control cells labeled poorly with IN-1: only 23% of class II–containing compartments also stained for Ii chain cytoplasmic tail (Fig. 7 A). Similarly, the majority of these class II–containing vesicles showed no or little lgp staining under normal conditions (Fig. 7 A). In contrast, after leupeptin treatment, the number of structures containing both class II and Ii chain, or class II and lgp-B, was greatly increased, accounting for >70% of the total intracellular class II– positive structures (Fig. 7, A and B). These results confirmed that leupeptin induced the intracellular accumulation of Ii-αβ complexes and illustrated the redistribution of MHC class II molecules to lysosomes. Thus, leupeptininduced alterations in Ii chain cleavage would appear to induce class II transport to lysosomes and appearance of MIICs in cells that do not normally accumulate class II in lysosomal compartments.

Bottom Line: Major histocompatibility complex class II molecules are synthesized as a nonameric complex consisting of three alpha beta dimers associated with a trimer of invariant (Ii) chains.Our results suggest that alterations in the rate or efficiency of Ii chain processing can alter the postendosomal sorting of class II molecules, resulting in the increased accumulation of alpha beta dimers in lysosome-like MIIC.Thus, simple differences in Ii chain processing may account for the highly variable amounts of class II found in lysosomal compartments of different cell types or at different developmental stages.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Section de Recherche Institut National de la Santé et de la Recherche Médicale CJF-95.01, Paris, France.

ABSTRACT
Major histocompatibility complex class II molecules are synthesized as a nonameric complex consisting of three alpha beta dimers associated with a trimer of invariant (Ii) chains. After exiting the TGN, a targeting signal in the Ii chain cytoplasmic domain directs the complex to endosomes where Ii chain is proteolytically processed and removed, allowing class II molecules to bind antigenic peptides before reaching the cell surface. Ii chain dissociation and peptide binding are thought to occur in one or more postendosomal sites related either to endosomes (designated CIIV) or to lysosomes (designated MIIC). We now find that in addition to initially targeting alpha beta dimers to endosomes, Ii chain regulates the subsequent transport of class II molecules. Under normal conditions, murine A20 B cells transport all of their newly synthesized class II I-A(b) alpha beta dimers to the plasma membrane with little if any reaching lysosomal compartments. Inhibition of Ii processing by the cysteine/serine protease inhibitor leupeptin, however, blocked transport to the cell surface and caused a dramatic but selective accumulation of I-A(b) class II molecules in lysosomes. In leupeptin, I-A(b) dimers formed stable complexes with a 10-kD NH2-terminal Ii chain fragment (Ii-p10), normally a transient intermediate in Ii chain processing. Upon removal of leupeptin, Ii-p10 was degraded and released, I-A(b) dimers bound antigenic peptides, and the peptide-loaded dimers were transported slowly from lysosomes to the plasma membrane. Our results suggest that alterations in the rate or efficiency of Ii chain processing can alter the postendosomal sorting of class II molecules, resulting in the increased accumulation of alpha beta dimers in lysosome-like MIIC. Thus, simple differences in Ii chain processing may account for the highly variable amounts of class II found in lysosomal compartments of different cell types or at different developmental stages.

Show MeSH
Related in: MedlinePlus