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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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Selective redistribution of newly synthesized MHC  class II molecules to lysosomes in leupeptin-treated cells analyzed by Percoll gradient centrifugation. (A) Leupeptin does not  affect the density of compartments containing β-hexosaminidase  and Tfn-HRP, markers of lysosomes and endosomes, respectively. After a 3-h incubation with or without 2 mM leupeptin,  cells were permitted to internalize Tfn coupled to HRP for 30  min at 37°C (in the presence or absence of leupeptin) before homogenization and fractionation on Percoll density gradients. The  β-hexosaminidase and HRP activities were determined in individual fractions. High density fractions (bottom of the gradient)  contained most of the β-hexosaminidase activity, while Tfn-HRP  was mainly present in low density fractions. No difference was  observed between leupeptin-treated and control cells. (B) Redistribution of newly synthesized MHC class II molecules into high  density fractions in leupeptin-treated cells. After a pulse of  [35S]methionine and a 1-, 2-, or 4-h chase in the absence (left) or  presence (right) of leupeptin, I-Ab–expressing A20 cells were  fractionated on Percoll gradients. The membranes in each of the  gradient fractions were pelleted by centrifugation, lysed in Triton  X-100, and then immunoprecipitated using mAbs to I-Ab (Y3P).  The samples were analyzed by SDS-PAGE, and bands corresponding to class II β chains were quantified by phosphorimaging  and optical densitometry. Ii-p10 intensity was also quantified after the 4-h chase in the presence of leupeptin (bottom left). Leupeptin caused a strong redistribution of class II into high density fractions. The majority of Ii-p10 was also found in lysosome-containing fractions. As expected, Ii-p10 was barely detectable in control cells  and thus was not shown.
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Figure 5: Selective redistribution of newly synthesized MHC class II molecules to lysosomes in leupeptin-treated cells analyzed by Percoll gradient centrifugation. (A) Leupeptin does not affect the density of compartments containing β-hexosaminidase and Tfn-HRP, markers of lysosomes and endosomes, respectively. After a 3-h incubation with or without 2 mM leupeptin, cells were permitted to internalize Tfn coupled to HRP for 30 min at 37°C (in the presence or absence of leupeptin) before homogenization and fractionation on Percoll density gradients. The β-hexosaminidase and HRP activities were determined in individual fractions. High density fractions (bottom of the gradient) contained most of the β-hexosaminidase activity, while Tfn-HRP was mainly present in low density fractions. No difference was observed between leupeptin-treated and control cells. (B) Redistribution of newly synthesized MHC class II molecules into high density fractions in leupeptin-treated cells. After a pulse of [35S]methionine and a 1-, 2-, or 4-h chase in the absence (left) or presence (right) of leupeptin, I-Ab–expressing A20 cells were fractionated on Percoll gradients. The membranes in each of the gradient fractions were pelleted by centrifugation, lysed in Triton X-100, and then immunoprecipitated using mAbs to I-Ab (Y3P). The samples were analyzed by SDS-PAGE, and bands corresponding to class II β chains were quantified by phosphorimaging and optical densitometry. Ii-p10 intensity was also quantified after the 4-h chase in the presence of leupeptin (bottom left). Leupeptin caused a strong redistribution of class II into high density fractions. The majority of Ii-p10 was also found in lysosome-containing fractions. As expected, Ii-p10 was barely detectable in control cells and thus was not shown.

Mentions: To determine if the lgp/lamp-positive vesicles were lysosomes or late endosomes, we analyzed the distribution of I-Ab class II molecules by centrifugation in 27% Percoll gradients, which separate low density early and late endosomes from heavy density lysosomes. Metabolically labeled cells were chased for 1–4 h in the presence or absence of leupeptin, homogenized, and centrifuged in Percoll, and then individual fractions were monitored for marker enzymes and for MHC class II. As shown in Fig. 5 A, leupeptin had no effect on the sedimentation of lysosomes (β-hexosaminidase) that were recovered from the same heavy density fractions in control and treated cells (left). Similarly, conjugates of Tfn-HRP, a marker of the receptor recycling pathway (internalized for 30 min before cell homogenization), sedimented in the low density regions of the gradient both in the presence and absence of leupeptin (Fig. 5 A, right). Similar results were obtained for the early and late endosomal markers rab5 and rab7, respectively (not shown). Thus, leupeptin did not cause an overall shift of endosomal markers to heavy density lysosomes or an alteration in the density properties of lysosomes themselves.


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)

Selective redistribution of newly synthesized MHC  class II molecules to lysosomes in leupeptin-treated cells analyzed by Percoll gradient centrifugation. (A) Leupeptin does not  affect the density of compartments containing β-hexosaminidase  and Tfn-HRP, markers of lysosomes and endosomes, respectively. After a 3-h incubation with or without 2 mM leupeptin,  cells were permitted to internalize Tfn coupled to HRP for 30  min at 37°C (in the presence or absence of leupeptin) before homogenization and fractionation on Percoll density gradients. The  β-hexosaminidase and HRP activities were determined in individual fractions. High density fractions (bottom of the gradient)  contained most of the β-hexosaminidase activity, while Tfn-HRP  was mainly present in low density fractions. No difference was  observed between leupeptin-treated and control cells. (B) Redistribution of newly synthesized MHC class II molecules into high  density fractions in leupeptin-treated cells. After a pulse of  [35S]methionine and a 1-, 2-, or 4-h chase in the absence (left) or  presence (right) of leupeptin, I-Ab–expressing A20 cells were  fractionated on Percoll gradients. The membranes in each of the  gradient fractions were pelleted by centrifugation, lysed in Triton  X-100, and then immunoprecipitated using mAbs to I-Ab (Y3P).  The samples were analyzed by SDS-PAGE, and bands corresponding to class II β chains were quantified by phosphorimaging  and optical densitometry. Ii-p10 intensity was also quantified after the 4-h chase in the presence of leupeptin (bottom left). Leupeptin caused a strong redistribution of class II into high density fractions. The majority of Ii-p10 was also found in lysosome-containing fractions. As expected, Ii-p10 was barely detectable in control cells  and thus was not shown.
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Figure 5: Selective redistribution of newly synthesized MHC class II molecules to lysosomes in leupeptin-treated cells analyzed by Percoll gradient centrifugation. (A) Leupeptin does not affect the density of compartments containing β-hexosaminidase and Tfn-HRP, markers of lysosomes and endosomes, respectively. After a 3-h incubation with or without 2 mM leupeptin, cells were permitted to internalize Tfn coupled to HRP for 30 min at 37°C (in the presence or absence of leupeptin) before homogenization and fractionation on Percoll density gradients. The β-hexosaminidase and HRP activities were determined in individual fractions. High density fractions (bottom of the gradient) contained most of the β-hexosaminidase activity, while Tfn-HRP was mainly present in low density fractions. No difference was observed between leupeptin-treated and control cells. (B) Redistribution of newly synthesized MHC class II molecules into high density fractions in leupeptin-treated cells. After a pulse of [35S]methionine and a 1-, 2-, or 4-h chase in the absence (left) or presence (right) of leupeptin, I-Ab–expressing A20 cells were fractionated on Percoll gradients. The membranes in each of the gradient fractions were pelleted by centrifugation, lysed in Triton X-100, and then immunoprecipitated using mAbs to I-Ab (Y3P). The samples were analyzed by SDS-PAGE, and bands corresponding to class II β chains were quantified by phosphorimaging and optical densitometry. Ii-p10 intensity was also quantified after the 4-h chase in the presence of leupeptin (bottom left). Leupeptin caused a strong redistribution of class II into high density fractions. The majority of Ii-p10 was also found in lysosome-containing fractions. As expected, Ii-p10 was barely detectable in control cells and thus was not shown.
Mentions: To determine if the lgp/lamp-positive vesicles were lysosomes or late endosomes, we analyzed the distribution of I-Ab class II molecules by centrifugation in 27% Percoll gradients, which separate low density early and late endosomes from heavy density lysosomes. Metabolically labeled cells were chased for 1–4 h in the presence or absence of leupeptin, homogenized, and centrifuged in Percoll, and then individual fractions were monitored for marker enzymes and for MHC class II. As shown in Fig. 5 A, leupeptin had no effect on the sedimentation of lysosomes (β-hexosaminidase) that were recovered from the same heavy density fractions in control and treated cells (left). Similarly, conjugates of Tfn-HRP, a marker of the receptor recycling pathway (internalized for 30 min before cell homogenization), sedimented in the low density regions of the gradient both in the presence and absence of leupeptin (Fig. 5 A, right). Similar results were obtained for the early and late endosomal markers rab5 and rab7, respectively (not shown). Thus, leupeptin did not cause an overall shift of endosomal markers to heavy density lysosomes or an alteration in the density properties of lysosomes themselves.

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