Week 2 is complete and as promised in the previous blog post, here are some details for the upcoming project .
To begin, I will give you some background. In cells, things are always in motion, whether it is RNA leaving the nucleus or proteins transferring from the endoplasmic reticulum (ER) to the Golgi apparatus (Golgi), or vise versa. For this particular project, we are interested in the forward trafficking, i.e. from the ER to the Golgi. This forward trafficking pathway is mediated by the Coat protein complex II (COPII). COPII has five major components:Sar1, Sec13, Sec23, Sec24, and Sec31. Additionally, posttranslational modification plays an important role in the regulation of the COPII complex. Recently, our lab and others have found that O-linked β-N-acetylglucosamine (O-GlcNAc), a single sugar modification added to serine and threonine residues of intracellular proteins, decorates many COPII components.
Now for the reveal of my project! I will look at O-GlcNAcylation of Sec24D, a cargo-binding subunit of the COPII complex, because in humans, Sec24D mutations cause a subtype of osteogenesis imperfecta (also known as brittle bone disease), a collagen trafficking disorder. I will do this by answering the question, “What role does Sec24D O-GlcNAcylation play in collagen secretion?”
Here is how I hope to answer the question. I will first create a Sec24D knockout cell line using CRISPR-Cas9 genome editing. Then I will express unglycosylatable Sec24D mutants in the Sec24D knockout cells. Finally, I will determine the role of Sec24D O-GlcNAcylation in collagen secretion by using immunofluorescence microscopy and measuring collagen trafficking to the Golgi.
Here is what I think will happen after this project is complete. I believe that mutations in Sec24D O-GlcNAc sites will cause collagen secretion to decrease. By doing so, I hope to enhance our understanding of the impacts of O-GlcNAcylation on the function of Sec24D in collagen secretion and allow for future questions on the impacts of O-GlcNAc on other COPII proteins.
Also, check out this paper similar to our project but on Sec23 from our lab. It is filled with additional background information, and methods that I will be using.
This has been a very exciting two weeks, and I am looking forward to the next six weeks!
On next weeks blog we will have a celebrity interview, so please check it out.
