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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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Fractionation of leupeptin-treated A20 cells by free  flow electrophoresis. (A) A20 cells were pulse labeled for 20 min  and chased for 2 h in the presence of leupeptin before fractionation by FFE. Membranes collected in each fraction were pelleted by centrifugation and lysed in Triton X-100, and I-Ab class  II molecules were immunoprecipitated using mAb MKD6. The  samples were then analyzed by SDS-PAGE without boiling. The  positions of compact dimers (“C”), α, β chains, Ii-p10, and a p12  protein of unknown origin are indicated relative to the positions  of markers for the major protein peak (plasma membrane), endosomes/lysosomes (β-hexosaminidase), and anodally shifted CIIVcontaining fractions. (Left) Anode; (right) cathode. (B) I-Ab– expressing A20 cells were pulse labeled for 20 min and chased for  4 h in the presence of leupeptin before fractionation by FFE. The  positions of p70 (Ii-p10–αβ complexes), 60-kD peptide-loaded  compact dimers, free α and β chains, and Ii-p10 are indicated.  (C) Positions of marker enzymes for plasma membrane (alkaline  phosphodiesterase) and endosomes/lysosomes (β-hexosaminidase)  in the FFE profile shown in B. p70 codistributed largely with the  lysosomal marker β-hexosaminidase.
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Figure 3: Fractionation of leupeptin-treated A20 cells by free flow electrophoresis. (A) A20 cells were pulse labeled for 20 min and chased for 2 h in the presence of leupeptin before fractionation by FFE. Membranes collected in each fraction were pelleted by centrifugation and lysed in Triton X-100, and I-Ab class II molecules were immunoprecipitated using mAb MKD6. The samples were then analyzed by SDS-PAGE without boiling. The positions of compact dimers (“C”), α, β chains, Ii-p10, and a p12 protein of unknown origin are indicated relative to the positions of markers for the major protein peak (plasma membrane), endosomes/lysosomes (β-hexosaminidase), and anodally shifted CIIVcontaining fractions. (Left) Anode; (right) cathode. (B) I-Ab– expressing A20 cells were pulse labeled for 20 min and chased for 4 h in the presence of leupeptin before fractionation by FFE. The positions of p70 (Ii-p10–αβ complexes), 60-kD peptide-loaded compact dimers, free α and β chains, and Ii-p10 are indicated. (C) Positions of marker enzymes for plasma membrane (alkaline phosphodiesterase) and endosomes/lysosomes (β-hexosaminidase) in the FFE profile shown in B. p70 codistributed largely with the lysosomal marker β-hexosaminidase.

Mentions: We previously found that in leupeptin-treated A20 cells, complexes of Ii-p10 and I-Ad dimers selectively accumulated in CIIV, a vesicle population that can be resolved from all other intracellular organelles by FFE (Amigorena et al., 1995). The intracellular sites of accumulation of I-Ad and I-Ab were compared by fractionating leupeptin-treated cells using FFE. The distribution of I-Ad molecules was determined after a 20-min pulse with [35S]methionine and 2 h of chase in the presence of leupeptin. These conditions maximized the amount of I-Ad complexes that accumulate intracellularly because of leupeptin (Amigorena et al., 1995). As shown in Fig. 3 A, most Ii chain free I-Ad molecules (immunoprecipitated using mAb MKD6) were found in the nonshifted FFE fractions containing the bulk of cellular membranes including the plasma membrane. A second peak of class II was also detected in anodally shifted fractions corresponding to CIIV. Little I-Ad was detected in β-hexosaminidase–containing fractions, demonstrating that endosomes and lysosomes were not a major site of I-Ad accumulation after leupeptin treatment. The amounts of labeled I-Ad–bound Ii-p10 were too low to allow detection in FFE fractions (see above). As found previously, an unknown I-Ad–associated 12-kD protein was present at the plasma membrane and in FFE unshifted fractions, but this protein was not Ii chain derived (Amigorena et al., 1995). When the cells were fractionated after a 4 h chase in the presence of leupeptin or a 2-h chase in the absence of leupeptin, no I-Ad was detected in any FFE-shifted fraction (not shown; Amigorena et al., 1995).


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)

Fractionation of leupeptin-treated A20 cells by free  flow electrophoresis. (A) A20 cells were pulse labeled for 20 min  and chased for 2 h in the presence of leupeptin before fractionation by FFE. Membranes collected in each fraction were pelleted by centrifugation and lysed in Triton X-100, and I-Ab class  II molecules were immunoprecipitated using mAb MKD6. The  samples were then analyzed by SDS-PAGE without boiling. The  positions of compact dimers (“C”), α, β chains, Ii-p10, and a p12  protein of unknown origin are indicated relative to the positions  of markers for the major protein peak (plasma membrane), endosomes/lysosomes (β-hexosaminidase), and anodally shifted CIIVcontaining fractions. (Left) Anode; (right) cathode. (B) I-Ab– expressing A20 cells were pulse labeled for 20 min and chased for  4 h in the presence of leupeptin before fractionation by FFE. The  positions of p70 (Ii-p10–αβ complexes), 60-kD peptide-loaded  compact dimers, free α and β chains, and Ii-p10 are indicated.  (C) Positions of marker enzymes for plasma membrane (alkaline  phosphodiesterase) and endosomes/lysosomes (β-hexosaminidase)  in the FFE profile shown in B. p70 codistributed largely with the  lysosomal marker β-hexosaminidase.
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Related In: Results  -  Collection

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Figure 3: Fractionation of leupeptin-treated A20 cells by free flow electrophoresis. (A) A20 cells were pulse labeled for 20 min and chased for 2 h in the presence of leupeptin before fractionation by FFE. Membranes collected in each fraction were pelleted by centrifugation and lysed in Triton X-100, and I-Ab class II molecules were immunoprecipitated using mAb MKD6. The samples were then analyzed by SDS-PAGE without boiling. The positions of compact dimers (“C”), α, β chains, Ii-p10, and a p12 protein of unknown origin are indicated relative to the positions of markers for the major protein peak (plasma membrane), endosomes/lysosomes (β-hexosaminidase), and anodally shifted CIIVcontaining fractions. (Left) Anode; (right) cathode. (B) I-Ab– expressing A20 cells were pulse labeled for 20 min and chased for 4 h in the presence of leupeptin before fractionation by FFE. The positions of p70 (Ii-p10–αβ complexes), 60-kD peptide-loaded compact dimers, free α and β chains, and Ii-p10 are indicated. (C) Positions of marker enzymes for plasma membrane (alkaline phosphodiesterase) and endosomes/lysosomes (β-hexosaminidase) in the FFE profile shown in B. p70 codistributed largely with the lysosomal marker β-hexosaminidase.
Mentions: We previously found that in leupeptin-treated A20 cells, complexes of Ii-p10 and I-Ad dimers selectively accumulated in CIIV, a vesicle population that can be resolved from all other intracellular organelles by FFE (Amigorena et al., 1995). The intracellular sites of accumulation of I-Ad and I-Ab were compared by fractionating leupeptin-treated cells using FFE. The distribution of I-Ad molecules was determined after a 20-min pulse with [35S]methionine and 2 h of chase in the presence of leupeptin. These conditions maximized the amount of I-Ad complexes that accumulate intracellularly because of leupeptin (Amigorena et al., 1995). As shown in Fig. 3 A, most Ii chain free I-Ad molecules (immunoprecipitated using mAb MKD6) were found in the nonshifted FFE fractions containing the bulk of cellular membranes including the plasma membrane. A second peak of class II was also detected in anodally shifted fractions corresponding to CIIV. Little I-Ad was detected in β-hexosaminidase–containing fractions, demonstrating that endosomes and lysosomes were not a major site of I-Ad accumulation after leupeptin treatment. The amounts of labeled I-Ad–bound Ii-p10 were too low to allow detection in FFE fractions (see above). As found previously, an unknown I-Ad–associated 12-kD protein was present at the plasma membrane and in FFE unshifted fractions, but this protein was not Ii chain derived (Amigorena et al., 1995). When the cells were fractionated after a 4 h chase in the presence of leupeptin or a 2-h chase in the absence of leupeptin, no I-Ad was detected in any FFE-shifted fraction (not shown; Amigorena et al., 1995).

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