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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.

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Reversal of the leupeptin block results in MHC class II transport from  lysosomes to the cell surface. (A) Reversibility of the effect of leupeptin on the intracellular retention of I-Ab class II molecules. Cells were metabolically labeled for  20 min, and then chased in the continous presence of leupeptin for 4 h. Leupeptin  was then removed, and the cells were incubated for 0–24 h. After cell surface biotinylation, the cells were lysed, and total (left) or surface (right) I-Ab molecules  were immunoprecipitated sequentially. Upon removal of leupeptin, the total  amount of p70 (the Ii-p10–αβ complex) slowly decreased, while the amount of  peptide-loaded, SDS-stable compact dimers (“C”) increased. Ii-p10 completely  disappeared over this time course. After a lag (see B), compact dimers began to  appear at the cell surface. (B) Kinetics of compact dimer formation and transport  to the cell surface. Bands corresponding to total and surface compact dimers were  quantified by phosphorimaging. The lag between formation of compact dimers  and their subsequent appearance at the cell surface was 3–4 h.
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Figure 8: Reversal of the leupeptin block results in MHC class II transport from lysosomes to the cell surface. (A) Reversibility of the effect of leupeptin on the intracellular retention of I-Ab class II molecules. Cells were metabolically labeled for 20 min, and then chased in the continous presence of leupeptin for 4 h. Leupeptin was then removed, and the cells were incubated for 0–24 h. After cell surface biotinylation, the cells were lysed, and total (left) or surface (right) I-Ab molecules were immunoprecipitated sequentially. Upon removal of leupeptin, the total amount of p70 (the Ii-p10–αβ complex) slowly decreased, while the amount of peptide-loaded, SDS-stable compact dimers (“C”) increased. Ii-p10 completely disappeared over this time course. After a lag (see B), compact dimers began to appear at the cell surface. (B) Kinetics of compact dimer formation and transport to the cell surface. Bands corresponding to total and surface compact dimers were quantified by phosphorimaging. The lag between formation of compact dimers and their subsequent appearance at the cell surface was 3–4 h.

Mentions: After 4 h in leupeptin, most of the I-Ab molecules were, as expected, present as αβ–Ii-p10 complexes that migrated as 70-kD complexes (p70) when not boiled, but as monomeric α, β, and Ii-p10 chains after boiling (Fig. 8 A, time 0; NB and B, respectively). Some of the class II was also present as 60-kD peptide-loaded compact dimers (“C”) not associated with Ii-p10. Relatively little class II was transported to the cell surface (Fig. 8 A, time 0, righthand panels).


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)

Reversal of the leupeptin block results in MHC class II transport from  lysosomes to the cell surface. (A) Reversibility of the effect of leupeptin on the intracellular retention of I-Ab class II molecules. Cells were metabolically labeled for  20 min, and then chased in the continous presence of leupeptin for 4 h. Leupeptin  was then removed, and the cells were incubated for 0–24 h. After cell surface biotinylation, the cells were lysed, and total (left) or surface (right) I-Ab molecules  were immunoprecipitated sequentially. Upon removal of leupeptin, the total  amount of p70 (the Ii-p10–αβ complex) slowly decreased, while the amount of  peptide-loaded, SDS-stable compact dimers (“C”) increased. Ii-p10 completely  disappeared over this time course. After a lag (see B), compact dimers began to  appear at the cell surface. (B) Kinetics of compact dimer formation and transport  to the cell surface. Bands corresponding to total and surface compact dimers were  quantified by phosphorimaging. The lag between formation of compact dimers  and their subsequent appearance at the cell surface was 3–4 h.
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Related In: Results  -  Collection

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Figure 8: Reversal of the leupeptin block results in MHC class II transport from lysosomes to the cell surface. (A) Reversibility of the effect of leupeptin on the intracellular retention of I-Ab class II molecules. Cells were metabolically labeled for 20 min, and then chased in the continous presence of leupeptin for 4 h. Leupeptin was then removed, and the cells were incubated for 0–24 h. After cell surface biotinylation, the cells were lysed, and total (left) or surface (right) I-Ab molecules were immunoprecipitated sequentially. Upon removal of leupeptin, the total amount of p70 (the Ii-p10–αβ complex) slowly decreased, while the amount of peptide-loaded, SDS-stable compact dimers (“C”) increased. Ii-p10 completely disappeared over this time course. After a lag (see B), compact dimers began to appear at the cell surface. (B) Kinetics of compact dimer formation and transport to the cell surface. Bands corresponding to total and surface compact dimers were quantified by phosphorimaging. The lag between formation of compact dimers and their subsequent appearance at the cell surface was 3–4 h.
Mentions: After 4 h in leupeptin, most of the I-Ab molecules were, as expected, present as αβ–Ii-p10 complexes that migrated as 70-kD complexes (p70) when not boiled, but as monomeric α, β, and Ii-p10 chains after boiling (Fig. 8 A, time 0; NB and B, respectively). Some of the class II was also present as 60-kD peptide-loaded compact dimers (“C”) not associated with Ii-p10. Relatively little class II was transported to the cell surface (Fig. 8 A, time 0, righthand panels).

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.

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Related in: MedlinePlus