Functional characterization of a lysosomal sorting motif in the cytoplasmic tail of HLA-DObeta.
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 beta 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-DObeta 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 DObeta 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 DObeta-encoded motif is involved in the fine-tuning of the intracellular sorting.
Brunet
A
A
Laboratoire d'Immunologie Moléculaire, Département de Microbiologie et Immunologie, Université de Montréal, Québec H3C 3J7, Canada.
Samaan
A
A
Deshaies
F
F
Kindt
T J
TJ
Thibodeau
J
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016384:Consensus Sequence; D003593:Cytoplasm / Q000378:metabolism*; D019281:Dimerization; D006681:HLA-D Antigens / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D006681:HLA-D Antigens; C084245:HLA-DM antigens; C046148:HLA-DO antigens; D011993:Recombinant Fusion Proteins; D014443:Tyrosine