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From their synthesis to their cell surface display, MHC-Is travel to many compartments, including the endoplasmic reticulum, Golgi, and endosomes. They come close to a plethora of molecules, some of which regulate directly or indirectly their folding and trafficking. While many of these proteins are well characterized, such as those found in the peptide loading complex, others remain to be discovered. The proxiome can be studied using proximity labeling assays, such as BioID, which relies on a biotin ligase fused to a bait of interest that biotinylates lysine residues on nearby proteins. These modified targets can then be purified and identified by mass spectrometry. By fusing BioID to the HLA-A2 cytoplasmic tail, we have applied this technique to the MHC-I antigen and identified 209 potential specific interactors in HEK293 cells, including PDZD8 (LYVAC) and MIA3 (Tango1). We knocked out MIA3 in HEK293 cells and measured an increase in the expression of MHC-Is, suggesting a role for this vesicle budding protein in the regulation of MHC-I trafficking. Notably, MHC-I crosslinking identified targets that connect reverse signaling to diverse metabolic processes. Altogether, our results underscore the promise of HLA-coupled biotin ligases as a powerful approach to dissect antigen presentation pathways.<\/jats:p>","DOI":"10.1093\/jleuko\/qiag035","type":"journal-article","created":{"date-parts":[[2026,3,14]],"date-time":"2026-03-14T12:37:24Z","timestamp":1773491844000},"source":"Crossref","is-referenced-by-count":0,"title":["A biotin ligation assay reveals a complex proxiome for HLA-A2 and implicates MIA3 in cell surface expression of MHC class I molecules"],"prefix":"10.1093","volume":"118","author":[{"given":"William","family":"Mitchell","sequence":"first","affiliation":[{"name":"D\u00e9partement de Microbiologie, Infectiologie et Immunologie, Laboratoire d\u2019Immunologie Mol\u00e9culaire, Universit\u00e9 de Montr\u00e9al , Montr\u00e9al, Qu\u00e9bec ,","place":["Canada"]},{"name":"Facult\u00e9 de M\u00e9decine, Institut Courtois d' innovation biom\u00e9dicale (CI2B), Universit\u00e9 de Montr\u00e9al , Montr\u00e9al, Qu\u00e9bec 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