Proteomic Analysis of Regulated Dendritic Cell Endosomes Reveals Dynamic Adaptation to Antigen Uptake and Cross‐Presentation

ABSTRACT MHC class I (MHC‐I) cross‐presentation involves final proteolytic peptide processing by the endosomal insulin‐regulated aminopeptidase (IRAP). Reasoning that analysis of the IRAP‐proximal proteome may inform about dynamic remodeling of a key cross‐presentation compartment during antigen uptake, we developed a proximity biotinylation system by expressing an IRAP‐TurboID fusion protein in MuTuDCs, a cell line resembling murine type 1 conventional dendritic cells. Analysis of luminal proteins associated with IRAP at steady state and during phagocytosis revealed a massive shift upon uptake of yeast but not apoptotic cells, favoring enrichment of antigen‐processing machinery, MHC‐I molecules, and proteins involved in ER‐associated folding and trafficking. Importantly, Sec22b modulated this proteomic landscape, promoting the localization of MHC‐I cross‐presentation proteins (e.g., MHC‐I, Tap1, Wdfy4, transferrin receptor) to the IRAP environment, while its absence favored MHC‐II and ER‐related proteins, suggesting a Sec22b‐dependent dichotomy between pathways favoring cross‐presentation versus antigen degradation. Intriguingly, uptake of apoptotic cells failed to promote cross‐presentation but induced two proteins related to immune tolerance, suggesting potential adaptation of proteome modulation to the outcome of antigen presentation. These data suggest that IRAP + endosomes serve as adaptive hubs integrating secretory and endocytic pathways, with Sec22b acting as a key determinant in tailoring this compartment for MHC‐I‐mediated cross‐presentation.