Regulation of Sec24 by O-GlcNAcylation

Coat protein complex II (COPII) mediates forward vesicle trafficking from the endoplasmic reticulum (ER). As an evolutionarily conserved system, COPII is essential for maintaining the lipid and protein composition of cellular membranes. COPII consists of five proteins essential for in vitro vesicle formation: Sar1, Sec23, Sec24, Sec13, and Sec31. Beautiful biochemical, genetic, and structural studies provided many insights into the function and geometry of the COPII coat. However, how COPII is regulated in response to environmental or cellular stimuli remains under-explored.

Like other proteins, COPII components are extensively post-translationally modified (PTM), which may function to modulate COPII activity. Of particular interest is O-linked β-N-acetylglucosamine (O-GlcNAc), a nutrient-sensitive modification of serine and threonine residues of nuclear, cytoplasmic, and mitochondrial proteins. Recently, we and others have found extensive O-GlcNAcylation of COPII components, including Sec23A, Sec24C, Sec24D, and Sec31A. To investigate the role of O-GlcNAc on these proteins, we used mass spectrometry to unambiguously identify O-GlcNAc-modified sites. We then used CRISPR/Cas9 to delete Sec23A, Sec24C, Sec24D, and Sec31A from a variety of cell lines and re-expressed unglycosylatable Ser/ThrAla mutants. This work primarily focuses on Sec24C and Sec24D.

We found that Sec24C is dynamically O-GlcNAcylated on a timescale of minutes, suggesting that this modification plays a regulatory/signaling role, as opposed to a structural role. We also found that Sec24C O-GlcNAcylation may regulate protein-protein interactions. In particular, we discovered that unglycosylatable Sec24C mutants altered the interaction between Sec24C and the Ebola protein VP40, suggesting that Sec24C O-GlcNAcylation may influence Ebola virus replication.

We also investigated the effect of a variety of cellular stressors on Sec24C O-GlcNAcylation using immunoprecipitation and immunoblot. We found that Sec24C O-GlcNAcylation was increased with rapamycin, suggesting that autophagy may trigger Sec24C O-GlcNAcylation. However, autophagy induction via amino-acid starvation failed to produce the same effect, suggesting that Sec24C O-GlcNAcylation may be the result of other signaling by mTORC1/2.

Finally, we identified six novel O-GlcNAc sites on Sec24D, that may function in COPII trafficking. Because Sec24D is required for collagen trafficking, we are currently testing the functionality of unglycosylatable Sec24D mutants in both human cell lines and a vertebrate model of skeletogenesis.

Together, this work suggests that O-GlcNAcylation plays multifaced roles on Sec24C and Sec24D and may function to integrate COPII function with cellular metabolism. Future studies on site-specific O-GlcNAcylation of Sec24 proteins will aid in our understanding of COPII regulation and provide valuable insight into the complex web of protein and lipid trafficking.