Sorting of MHC class II molecules into exosomes through a ubiquitin-independent pathway.
Major histocompatibility complex class II (MHC-II) molecules accumulate in exocytic vesicles, called exosomes, which are secreted by antigen presenting cells. These vesicles are released following the fusion of multivesicular bodies (MVBs) with the plasma membrane. The molecular mechanisms regulating cargo selection remain to be fully characterized. As ubiquitination of the MHC-II beta-chain cytoplasmic tail has recently been demonstrated in various cell types, we sought to determine if this post-translational modification is required for the incorporation of MHC-II molecules into exosomes. First, we stably transfected HeLa cells with a chimeric HLA-DR molecule in which the beta-chain cytoplasmic tail is replaced by ubiquitin. Western blot analysis did not indicate preferential shedding of these chimeric molecules into exosomes. Next, we forced the ubiquitination of MHC-II in class II transactivator (CIITA)-expressing HeLa and HEK293 cells by transfecting the MARCH8 E3 ubiquitin ligase. Despite the almost complete downregulation of MHC-II from the plasma membrane, these molecules were not enriched in exosomes. Finally, site-directed mutagenesis of all cytoplasmic lysine residues on HLA-DR did not prevent inclusion into these vesicles. Taken together, these results demonstrate that ubiquitination of MHC-II is not a prerequisite for incorporation into exosomes.
Gauvreau
Marie-Elaine
ME
Laboratoire d'Immunologie Moléculaire, Département de Microbiologie et Immunologie, Université de Montréal, Québec, Canada.
Côté
Marie-Hélène
MH
Bourgeois-Daigneault
Marie-Claude
MC
Rivard
Louis-David
LD
Xiu
Fangming
F
Brunet
Alexandre
A
Shaw
Andrew
A
Steimle
Viktor
V
Thibodeau
Jacques
J
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism; D015536:Down-Regulation; D055354:Exosomes / Q000378:metabolism*; D005434:Flow Cytometry; D006684:HLA-DR Antigens / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases