Hirata, T.; Choudhary, D.; Bisnett, B. J.; Soderblom, E. J.; Knapik, E. W.; Boyce, M.

Coat protein complex II (COPII) mediates anterograde trafficking from the endoplasmic reticulum (ER). While the core COPII machinery is well-characterized, how cells regulate COPII to accommodate large cargoes, including collagens, remains incompletely understood. Here, we show that the cargo-selecting COPII subunit Sec24D is modified by site-specific O-linked {beta}-N-acetylglucosamine (O-GlcNAc) in its N-terminal intrinsically disordered region upon induction of collagen transport. These glycosylations are required for collagen trafficking in human cells and developing zebrafish. Crosslinking proteomics demonstrated that each O-GlcNAcylation influences the Sec24D interactome in a distinct way, revealing novel mediators of COPII function. In particular, Sec24D glycosylation is required for its interaction with myoferlin, which unexpectedly facilitates fusion of ER exit sites (ERES) and the ER- Golgi intermediate compartment (ERGIC) to enable collagen transport. Our results establish Sec24D O-GlcNAcylation as a dynamic regulator of COPII protein-protein interactions and collagen trafficking and identify myoferlin as a novel mediator of this process.