Functional characterization of a lysosomal sorting motif in the cytoplasmic tail of HLA-DO beta

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 molecu les such as HLA-DR. The activity of HLA-DO is mediated through its interact ion with the peptide editor HLA-DM. Here, our results demonstrate that alth ough HLA-DO is absolutely dependent on its association with DM: to egress t he endoplasmic reticulum, the cytoplasmic portion of its beta chain encodes a functional lysosomal sorting signal, By confocal microscopy and flow cyt ometry analysis, we show that reporter transmembrane molecules fused to the cytoplasmic tail of HLA-DO beta accumulated in Lamp-1(+) vesicles of trans fected HeLa cells. Mutagenesis of a leucine-leucine motif abrogated lysosom al accumulation and resulted in cell surface redistribution of reporter mol ecules. Finally, we show that mutation of the di-leucine sequence in DO bet a did not alter its lysosomal sorting when associated with DM molecules. Ta ken 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 beta -encoded motif is involved in the fine-tuning of the intracellular sorting.