Expression of Major Histocompatibility Complex Class II Molecules in HeLa Cells Promotes the Recruitment of AP-1 Golgi-specific Assembly Proteins on Golgi Membranes

Abstract

The newly synthesized major histocompatibility complex (MHC) class II molecules, an ␣␤ dimer associated with the Ii invariant chain, must be targeted to endosomal, lysosomal enzyme-rich compartments in order to bind and present immunogenic peptides. The precise route followed by this complex at the exit of the transGolgi network, the last sorting station of the biosynthetic pathway, is poorly understood. We show here that overexpression of ␣␤Ii complexes in HeLa cells promotes the first step of clathrin-coat assembly in vitro, that is the ARF-dependent translocation of AP-1 Golgispecific assembly proteins on membranes. In contrast, ␣␤ dimers alone or associated with Ii lacking most of its cytoplasmic domain fail to recruit AP-1. This study strongly suggests that the invariant chain (Ii) is responsible for the AP-1-dependent sorting of the ␣␤ dimers in the trans-Golgi network of HeLa cells and that the MHC class II molecules are, like the mannose 6-phosphate receptors, transported directly from this compartment to endosomes via clathrin-coated vesicles.The major histocompatibility complex (MHC) 1 class II molecules bind peptides produced by the proteolysis of foreign antigens in endocytic, hydrolase-rich compartments and mediate their presentation to competent T-lymphocytes (1-3). The ␣ and ␤ polypeptides that form the MHC class II complex associate with the invariant chain (Ii) in the endoplasmic reticulum. The whole complex is then transported through the biosynthetic pathway for delivery to endosomal/lysosomal compartments where Ii dissociates from ␣␤ dimers. The invariant chain contains sorting signals in its cytoplasmic domain that confer endosomal/lysosomal targeting to the MHC class II molecules (4 -6). While the fate of the MHC class II molecules in the early secretory and in the endocytic pathways has been described extensively, little is known about their vesicular transport at the exit of the trans-Golgi network. Whether the MHC-II molecules are targeted directly to endosomal compartments via clathrin-coated vesicles like lysosomal hydrolases bound to the mannose 6-phosphate receptors (MPRs) (7) or via other types of carrier vesicles, as recently proposed for I-cell disease B-lymphoblasts (8), has been difficult to evaluate.The first step in the formation of TGN-derived, clathrincoated vesicles is the translocation of the cytosolic AP-1 Golgispecific assembly proteins onto membranes of this compartment (9), a process regulated by the small GTPase ARF-1 (10, 11). We have shown previously that the mannose 6-phosphate receptors, the major cargo membrane proteins sorted along this clathrin-dependent pathway, are part of the membrane components that permit the efficient recruitment of AP-1 (12, 13), thereby strongly suggesting that MPR sorting is highly coupled to the first step of clathrin coat assembly. Moreover, efficient AP-1 binding to membranes requires the presence of specific determinants in the MPR cytoplasmic domains (14). The property that the expression of cargo transmembrane proteins triggers A...

Birbeck Granules Are Subdomains of Endosomal Recycling Compartment in Human Epidermal Langerhans Cells, Which Form Where Langerin Accumulates

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“…After fixation and permeabilization the cells were labeled simultaneously with an FITC-conjugated anti-CD1a mAb and phalloidine-TRITC, to study in LCs the effects of cytochalasin D and latrunculin A on the organization of actin filaments, or with CTR-433 followed by donkey anti-mouse TRITC and finally an FITC-conjugated anti-CD1a mAb to visualize in LCs the effects of BFA on Golgi distribution. Confocal laser scanning microscopy and immunofluorescence quantification were performed as previously described (Salamero et al, 1996) by using a Leica TCS4D confocal microscope (Leica Microsystems, Heidelberg, Germany). …”

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Early Embryonic Death of Mice Deficient in γ-Adaptin

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