Role of O‐GlcNAcylation in SEC24C Function

Coat protein complex II (COPII) mediates forward protein and lipid cargo trafficking from the endoplasmic reticulum via five protein components required for in vitro vesicle formation: SAR1, SEC13, SEC23, SEC24, and SEC31. While the structure and function of COPII proteins are well understood, we do not fully understand how COPII is regulated in response to environmental and cellular stimuli. We and others have found that O‐linked‐beta‐N‐acetylglucosamine (O‐GlcNAc), a dynamic single‐sugar modification added to serines and threonines of intracellular proteins, decorates many human COPII components, including all four SEC24 paralogs. Additionally, others have shown that COPII, and specifically SEC24, may play a role in the formation of the autophagosome for autophagy, a process in which the cell degrades its own components. However, the regulation of SEC24’s canonical and autophagic functions remains unclear. To determine the function of SEC24 O‐GlcNAcylation, we mapped O‐GlcNAc sites on SEC24C using mass spectrometry and created unglycosylatable serine or threonine to alanine mutations at each O‐GlcNAc site. We then used CRISPR‐Cas9 to create SEC24C−/− HEK 293T and HeLa cells, and reintroduced unglycosylatable SEC24C mutants. By comparing SEC24C−/− to control HeLa cells, we discovered a baseline difference between levels of autophagy. Furthermore, SEC24C−/− 293T cells may be more sensitive to ER stress than control cells are. With these differences between SEC24C −/− and control cells, we will next determine whether the differences persist when comparing the unglycosylatable mutants to wild type SEC24C, using Western blots and immunoprecipitation. This study will enhance our understanding of the role of O‐GlcNAcylation on SEC24C function and the interplay between canonical COPII function and autophagy.Support or Funding InformationResearch on this project in the Boyce Lab is supported by NIH grant 1R01GM117473.