HLA-DO is an intracellular non-classical class II major histocompatibility complex molecule expressed in the endocytic pathway of B lymphocytes, which regulates the loading of antigenic peptides onto classical class II molecules such as HLA-DR. The activity of HLA-DO is mediated through its interaction with the peptide editor HLA-DM. Here, our results demonstrate that although HLA-DO is absolutely dependent on its association with DM to egress the endoplasmic reticulum, the cytoplasmic portion of its  chain encodes a functional lysosomal sorting signal. By confocal microscopy and flow cytometry analysis, we show that reporter transmembrane molecules fused to the cytoplasmic tail of HLA-DO accumulated in Lamp-1 ؉ vesicles of transfected HeLa cells. Mutagenesis of a leucine-leucine motif abrogated lysosomal accumulation and resulted in cell surface redistribution of reporter molecules. Finally, we show that mutation of the di-leucine sequence in DO did not alter its lysosomal sorting when associated with DM molecules. Taken together, these results demonstrate that lysosomal expression of the DO-DM complex is mediated primarily by the tyrosine-based motif of HLA-DM and suggest that the DO-encoded motif is involved in the fine-tuning of the intracellular sorting. Major histocompatibility complex (MHC)1 class II molecules are heterodimeric cell surface glycoproteins that present antigens to CD4 ϩ T cells (1). The antigenic peptide-class II complexes are expressed on specialized antigen-presenting cells such as macrophages and B lymphocytes. Following their synthesis, the ␣ and  subunits of the class II molecule associate in the endoplasmic reticulum (ER) together with the invariant chain (Ii) (2). The latter folds in part through the groove of the class II molecule, stabilizing the ␣ heterodimer and preventing the undesirable binding of ER polypeptides (3-6). Studies using mice with inactivated Ii genes suggested that Ii is necessary for efficient exit of newly synthesized class II molecules from the ER (7, 8). However, it was later demonstrated that high levels of class II molecules reach the surface of Ii Ϫ dendritic cells (9). Moreover, transfected cells express a substantial amount of class II molecules at their plasma membrane even in the absence of Ii (10). Such a phenotype probably results from the binding of endogenous peptides or polypeptides present in the ER (6, 11) and supports the notion that occupancy of the peptide-binding groove is sufficient to allow ER egress (12).Another function of Ii is to direct efficiently MHC class II molecules to the endocytic antigen-loading compartments (13-15). Two short leucine-based sequences located in the cytoplasmic tail of Ii are responsible for trafficking through the endocytic pathway (16,17). Similar motifs in many proteins are specifically recognized at the cell surface and trans-Golgi by elements of the sorting machinery (reviewed in Ref. 18).Once the class II-Ii complex reaches the endosomal compartments, the invariant chain is progressively degraded by v...
B lymphocytes express the nonclassical class II molecule HLA-DO, which modulates the peptide loading activity of HLA-DM in the endocytic pathway. Binding to HLA-DM is required for HLA-DO to egress from the endoplasmic reticulum (ER). To gain insights into the mode of action of DO and on the role of DM in ER release, we sought to identify DM-binding residues on DO. Our results show that DO␣ encompasses the binding site for HLA-DM. More specifically, mutation of residue DO␣41 on an exposed lateral loop of the ␣1 domain affects the binding to DM, ER egress, and activity of DO. Using a series of chimeric DR͞DO molecules, we confirmed the role of the ␣ chain and established that a second DM-binding region is located C-terminal to the DO␣80 residue, most probably in the ␣2 domain. Interestingly, after mutation of a buried proline (␣11) on the floor of the putative peptide-binding groove, HLA-DO remained functional but became independent of HLA-DM for ER egress and intracellular trafficking. Collectively, these results suggest that the binding of HLA-DM to DO␣ allows the complex to egress from the ER by stabilizing intramolecular contacts between the N-terminal antiparallel -strands of the DO␣ heterodimer.antigen processing ͉ MHC ͉ class II ͉ HLA-DR C lassical MHC class II proteins are highly polymorphic heterodimers expressed at the surface of antigen-presenting cells where they bind antigenic peptides derived from endocytosed antigens (1). The ␣ and  subunits associate in the endoplasmic reticulum (ER) with the nonpolymorphic invariant chain (Ii), and the complex is sorted to endocytic compartments where Ii is degraded. A small fragment [class II-associated Ii peptide (CLIP)] of the Ii is protected inside the groove and must be released before the binding of antigenic peptides (2). HLA-DM (DM), a nonpolymorphic intracellular chaperone, is responsible for CLIP removal and also for editing the peptide repertoire to favor those of higher class II-binding affinity (3).Class II-restricted antigen presentation in B lymphocytes is tightly regulated to ensure specificity of the activation process. These cells express another nonclassical class II molecule called HLA-DO (DO; H2-O in mice) that modulates the presentation of antigens in the endocytic pathway (4). Recently, it was shown that transfection of DO in class II transactivator (CIITA) ϩ cells caused the accumulation of classical class II molecules associated with CLIP (5, 6). These results clearly showed the inhibitory role of DO on class II-restricted antigen presentation.The precise molecular mechanism by which DO inhibits the catalytic activity of DM remains to be clarified. DO is found mostly in endosomes but is retained in the ER of DM Ϫ cells. Interestingly, DO-bound DM is not sequestered there but rather allows the complex to egress from the ER (7). Trafficking of DO͞DM to and inside the endocytic pathway is regulated by sorting signals located in the cytoplasmic tails of both molecules (8, 9). The need for DO to access peptide-loading compartments and to mod...
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