To establish the impact of Sec24C O-GlcNAcylation on COPII secretion, I will use Sec24C CRISPR-deleted 293T human kidney cells (kindly provided by D. Ginsburg) and stably re-introduce wild type or unglycosylatable mutant (S98A, T98A, and T201A) Sec24C expression constructs. I will test these cells in four assays to evaluate COPII secretion. (1) I will use the secretion of horseradish peroxidase containing a COPII secretion signal (ssHRP) to quantitate secretion by assessing the accumulation of ssHRP in the medium 19. In addition to the secretion of HRP, I would like to test the reliance or large cargoes on Sec24C O-GlcNAcylation. Large cargos such as collagen rely on specific COPII paralogs as well as post-translational modifications of the COPII coat 9,20. I will make a Collagen 1A1-HRP fusion construct to examine requirement of Sec24C glycosylation for the transport of a large COPII cargo. (2) I will use fluorescence microscopy to look at the secretion of two Sec24C-dependent cargos: the serotonin transporter (SERT) 21 and an Ebola protein, VP40 22. (3) I will utilize a well-characterized Brefeldin A (BFA) recovery assay to measure the reconstitution of the Golgi after brief treatment with BFA. BFA is a fungal compound that causes the collapse of the Golgi through inhibition of the Arf1 GTPase 23,24. Inhibition of COPII function results in slowed reconstitution of the Golgi after BFA treatment. By monitoring the COPII-dependent trafficking of Golgi markers GOS-28 and giantin by immunofluorescence microscopy, I will assess the degree of Golgi reconstitution after BFA washout. (4) Lastly, I will test the membrane association of Sec24C using the cholesterol...
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...PII vesicle trafficking. Using a series of functional assays I will address the role O-GlcNAcylated Sec24C in the COPII pathway. I will also identify O-GlcNAc-mediated binding partners of Sec24C through GlcNDAz crosslinking. Finally I will examine the interplay between phosphorylation and O-GlcNAcylation on Sec24C. These experiments will provide insight into the regulation of COPII by PTMs and address a long-standing gap in knowledge in the COPII field. In addition, they may inform us about the regulation of other Sec24 paralogs by PTMs. Like Sec24C, Sec24B contains multiple O-GlcNAcylation sites on the N-terminal region 28,29. Future directions include investigation of the functional significance of O-GlcNAcylation on other Sec24 paralogs as well as the development of zebrafish models to examine developmental and tissue specific requirements Sec24C O-GlcNAcylation.
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