Specific Sites On Sec24c / D Regulate Copii Trafficking And Mediate Protein Proteins Interactions Essay
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I hypothesize that O-GlcNAcylation of specific sites on Sec24C/D regulate COPII vesicle trafficking and mediate protein-protein interactions. The experiments proposed below will test this hypothesis by completion of my three aims: 1) Determine how O-GlcNAcylation of Sec24C/D affects COPII vesicle secretion under normal and ER stress conditions, 2) Characterize Sec24C/D O-GlcNAc mediated protein-protein interactions and their role in vesicle trafficking and, 3) Examine the Interplay between O-GlcNAcylation and phosphorylation on Sec24C/D in cell cycle progression and vesicle trafficking.
Aim1: Determine how O-GlcNAcylation of Sec24C/D affects COPII vesicle secretion under normal and ER stress conditions. Research design: I previously identified nine O-GlcNAc sites on Sec24C. This was accomplished by expressing myc-6xHis tagged Sec24C in human embryonic kidney 293T cells treated with Thiamet-G, an OGA inhibitor, and glucosamine to increase O-GlcNAc occupancy. After isolating Sec24C through myc immunoprecipitation and Ni-NTA purification, the sample was submitted to the Duke proteomics and metabolomics facility. Using this same technique, I will identify O-GlcNAcylation sites on the closely related Sec24D. In collaboration with the Duke Functional Genomics Shared Resource, I will then make Sec24C or Sec24D loss of function HEK 293T, Hela and chondrocyte cell lines made with CRISPR-Cas9 technology. I will express unglycosylatable alanine mutants of Sec24C and Sec24D in