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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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Leupeptin induces  an accumulation of SDS- resistant I-Ab Ii-p10 complexes. (A) Leupeptin induces the accumulation of  10-kD (Ii-p10) and 70-kD  (p70) proteins that coprecipitate with I-Ab. I-Ab–expressing A20 cells were pulsed for  20 min with [35S]methionine  and chased at 37°C for the indicated times in the presence  or absence of 2 mM leupeptin. After lysis, the I-Ab molecules were immunoprecipitated using the Y3P mAb.  The samples were not boiled  before SDS-PAGE. Labeled  class II molecules were not  detected before 30 min of  chase because the Y3P mAb  used for immunoprecipitation does not detect immature αβ dimers complexed  with intact Ii chain. (B) p70  represents SDS-stable complexes containing class II α  and β chains and a 10-kD  protein. After a 20-min  pulse and 4-h chase with or  without 2 mM leupeptin  (lanes Lp and C, respectively), class II molecules  were immunoprecipitated using the Y3P mAb, and the  samples were boiled (B) or  not boiled (NB) before SDSPAGE. After boiling, p70  dissociated quantitatively  into monomers corresponding to αβ and Ii-p10. (C) p70  represents SDS-stable I-Ab  αβ–Ii-p10 complexes. After a 20-min pulse and 4-h chase in the presence of leupeptin, class II molecules were immunoprecipitated with  either anti–I-Ab (Y3P) or anti–Ii chain cytoplasmic domain (IN-1) mAbs. While both antibodies precipitated the p70 complex, only  anti–class II mAb precipitated the 60-kD SDS-stable compact dimer. Thus, p70 but not compact dimers are complexed with Ii chain or  Ii chain fragments (i.e., Ii-p10) that contain the Ii chain cytoplasmic domain. (D) Kinetics of association between Ii-p10 and I-Ab or  I-Ad. Pulse-chase experiments were performed as above using A20 cells expressing only I-Ad or expressing both I-Ad and I-Ab. I-Ad  or I-Ab–containing complexes were then immunoprecipitated using specific mAbs (Y3P and MKD6, respectively), and the amounts of  Ii-p10 associated to the class II molecules were quantified by phosphorimaging. The association of Ii-p10 with I-Ab persisted throughout the chase period, while Ii-p10–I-Ad complexes appeared only transiently.
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Figure 1: Leupeptin induces an accumulation of SDS- resistant I-Ab Ii-p10 complexes. (A) Leupeptin induces the accumulation of 10-kD (Ii-p10) and 70-kD (p70) proteins that coprecipitate with I-Ab. I-Ab–expressing A20 cells were pulsed for 20 min with [35S]methionine and chased at 37°C for the indicated times in the presence or absence of 2 mM leupeptin. After lysis, the I-Ab molecules were immunoprecipitated using the Y3P mAb. The samples were not boiled before SDS-PAGE. Labeled class II molecules were not detected before 30 min of chase because the Y3P mAb used for immunoprecipitation does not detect immature αβ dimers complexed with intact Ii chain. (B) p70 represents SDS-stable complexes containing class II α and β chains and a 10-kD protein. After a 20-min pulse and 4-h chase with or without 2 mM leupeptin (lanes Lp and C, respectively), class II molecules were immunoprecipitated using the Y3P mAb, and the samples were boiled (B) or not boiled (NB) before SDSPAGE. After boiling, p70 dissociated quantitatively into monomers corresponding to αβ and Ii-p10. (C) p70 represents SDS-stable I-Ab αβ–Ii-p10 complexes. After a 20-min pulse and 4-h chase in the presence of leupeptin, class II molecules were immunoprecipitated with either anti–I-Ab (Y3P) or anti–Ii chain cytoplasmic domain (IN-1) mAbs. While both antibodies precipitated the p70 complex, only anti–class II mAb precipitated the 60-kD SDS-stable compact dimer. Thus, p70 but not compact dimers are complexed with Ii chain or Ii chain fragments (i.e., Ii-p10) that contain the Ii chain cytoplasmic domain. (D) Kinetics of association between Ii-p10 and I-Ab or I-Ad. Pulse-chase experiments were performed as above using A20 cells expressing only I-Ad or expressing both I-Ad and I-Ab. I-Ad or I-Ab–containing complexes were then immunoprecipitated using specific mAbs (Y3P and MKD6, respectively), and the amounts of Ii-p10 associated to the class II molecules were quantified by phosphorimaging. The association of Ii-p10 with I-Ab persisted throughout the chase period, while Ii-p10–I-Ad complexes appeared only transiently.

Mentions: As shown in Fig. 1 A (left), control cells began to accumulate SDS-stable I-Ab compact dimers (“C”) within 60 min of chase. The amount of the 60-kD dimer reached a plateau by 120 min and remained constant, accounting for ∼90% of the transfected I-Ab recovered from these cells. This is in contrast with the endogenous αβ dimers of the I-Ad haplotype, a much smaller fraction of which (15%) can be rendered SDS stable by peptide binding (Germain and Hendrix, 1991; Amigorena et al., 1995). Little class II was immunoprecipitated at time points <60 min since the antibody used (Y3P) does not detect class II molecules bound to intact Ii chain. Using a polyclonal antibody that detects all forms of class II, equivalent recovery of class II was found at all chase times, indicating that there was no degradation during the chase period (0–240 min; not shown).


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)

Leupeptin induces  an accumulation of SDS- resistant I-Ab Ii-p10 complexes. (A) Leupeptin induces the accumulation of  10-kD (Ii-p10) and 70-kD  (p70) proteins that coprecipitate with I-Ab. I-Ab–expressing A20 cells were pulsed for  20 min with [35S]methionine  and chased at 37°C for the indicated times in the presence  or absence of 2 mM leupeptin. After lysis, the I-Ab molecules were immunoprecipitated using the Y3P mAb.  The samples were not boiled  before SDS-PAGE. Labeled  class II molecules were not  detected before 30 min of  chase because the Y3P mAb  used for immunoprecipitation does not detect immature αβ dimers complexed  with intact Ii chain. (B) p70  represents SDS-stable complexes containing class II α  and β chains and a 10-kD  protein. After a 20-min  pulse and 4-h chase with or  without 2 mM leupeptin  (lanes Lp and C, respectively), class II molecules  were immunoprecipitated using the Y3P mAb, and the  samples were boiled (B) or  not boiled (NB) before SDSPAGE. After boiling, p70  dissociated quantitatively  into monomers corresponding to αβ and Ii-p10. (C) p70  represents SDS-stable I-Ab  αβ–Ii-p10 complexes. After a 20-min pulse and 4-h chase in the presence of leupeptin, class II molecules were immunoprecipitated with  either anti–I-Ab (Y3P) or anti–Ii chain cytoplasmic domain (IN-1) mAbs. While both antibodies precipitated the p70 complex, only  anti–class II mAb precipitated the 60-kD SDS-stable compact dimer. Thus, p70 but not compact dimers are complexed with Ii chain or  Ii chain fragments (i.e., Ii-p10) that contain the Ii chain cytoplasmic domain. (D) Kinetics of association between Ii-p10 and I-Ab or  I-Ad. Pulse-chase experiments were performed as above using A20 cells expressing only I-Ad or expressing both I-Ad and I-Ab. I-Ad  or I-Ab–containing complexes were then immunoprecipitated using specific mAbs (Y3P and MKD6, respectively), and the amounts of  Ii-p10 associated to the class II molecules were quantified by phosphorimaging. The association of Ii-p10 with I-Ab persisted throughout the chase period, while Ii-p10–I-Ad complexes appeared only transiently.
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Related In: Results  -  Collection

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

Figure 1: Leupeptin induces an accumulation of SDS- resistant I-Ab Ii-p10 complexes. (A) Leupeptin induces the accumulation of 10-kD (Ii-p10) and 70-kD (p70) proteins that coprecipitate with I-Ab. I-Ab–expressing A20 cells were pulsed for 20 min with [35S]methionine and chased at 37°C for the indicated times in the presence or absence of 2 mM leupeptin. After lysis, the I-Ab molecules were immunoprecipitated using the Y3P mAb. The samples were not boiled before SDS-PAGE. Labeled class II molecules were not detected before 30 min of chase because the Y3P mAb used for immunoprecipitation does not detect immature αβ dimers complexed with intact Ii chain. (B) p70 represents SDS-stable complexes containing class II α and β chains and a 10-kD protein. After a 20-min pulse and 4-h chase with or without 2 mM leupeptin (lanes Lp and C, respectively), class II molecules were immunoprecipitated using the Y3P mAb, and the samples were boiled (B) or not boiled (NB) before SDSPAGE. After boiling, p70 dissociated quantitatively into monomers corresponding to αβ and Ii-p10. (C) p70 represents SDS-stable I-Ab αβ–Ii-p10 complexes. After a 20-min pulse and 4-h chase in the presence of leupeptin, class II molecules were immunoprecipitated with either anti–I-Ab (Y3P) or anti–Ii chain cytoplasmic domain (IN-1) mAbs. While both antibodies precipitated the p70 complex, only anti–class II mAb precipitated the 60-kD SDS-stable compact dimer. Thus, p70 but not compact dimers are complexed with Ii chain or Ii chain fragments (i.e., Ii-p10) that contain the Ii chain cytoplasmic domain. (D) Kinetics of association between Ii-p10 and I-Ab or I-Ad. Pulse-chase experiments were performed as above using A20 cells expressing only I-Ad or expressing both I-Ad and I-Ab. I-Ad or I-Ab–containing complexes were then immunoprecipitated using specific mAbs (Y3P and MKD6, respectively), and the amounts of Ii-p10 associated to the class II molecules were quantified by phosphorimaging. The association of Ii-p10 with I-Ab persisted throughout the chase period, while Ii-p10–I-Ad complexes appeared only transiently.
Mentions: As shown in Fig. 1 A (left), control cells began to accumulate SDS-stable I-Ab compact dimers (“C”) within 60 min of chase. The amount of the 60-kD dimer reached a plateau by 120 min and remained constant, accounting for ∼90% of the transfected I-Ab recovered from these cells. This is in contrast with the endogenous αβ dimers of the I-Ad haplotype, a much smaller fraction of which (15%) can be rendered SDS stable by peptide binding (Germain and Hendrix, 1991; Amigorena et al., 1995). Little class II was immunoprecipitated at time points <60 min since the antibody used (Y3P) does not detect class II molecules bound to intact Ii chain. Using a polyclonal antibody that detects all forms of class II, equivalent recovery of class II was found at all chase times, indicating that there was no degradation during the chase period (0–240 min; not shown).

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