Antigen presentation by major histocompatibility complex class I (MHC I) depends on the accurate assembly of peptide–MHC I complexes, yet the molecular mechanism coupling peptide loading to peptide-loading complex (PLC) disassembly remains unresolved. Progress has been limited by the lack of an in vitro system that faithfully recapitulates the native membrane environment. Here, we reconstitute intact human PLCs in liposomes, enabling analysis of peptide loading, MHC I release, and post-release glycan processing under membrane-reconstituted conditions. We show that high-affinity peptide binding triggers allomorph-specific release of MHC I from the PLC, independent of the conformational state of the transporter associated with antigen processing (TAP). Furthermore, glucosidase II-mediated N-glycan trimming and calreticulin dissociation occur predominantly after MHC I exits the PLC, revealing a sequential disassembly mechanism. These findings define the molecular sequence of events underlying PLC disassembly and provide a versatile platform for mechanistic studies of antigen processing.

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