Protein Degradation in Chloroplasts
Proteins are present in every facet of biological life and the timing of protein synthesis and degradation is key to ensure that each protein can perform its function effectively. To understand more about protein function and stability, we wanted to delve into how they are degraded, specifically in chloroplasts. The degradation pathway of interest is the N-end rule which states that certain N-terminal amino acids act as signals for degradation. N-end rule degradation pathways are present in both the cytosol of prokaryotes and eukaryotes. Our objective was to determine whether an N-end rule operates in chloroplasts and develop a versatile in planta system to test this idea. The end goal was to perform an in planta fluorescence assay using GFP and RFP reporter proteins to visualize degradation. The GFP would have a stabilizing N-terminal residue and the RFP would have a destabilizing N-terminal residue. The start of the degradation process would be controlled by using TEV2 protease, under inducible expression, to cleave the fluorescent reporters at a specific cleavage site and expose the chosen N-terminal residues. By monitoring the fluorescence intensity over time, we should be able to see the impact of specific N-terminal residues on protein degradation. We were able to successfully transform both the fluorescent proteins and TEV2 protease into Arabidopsis thaliana (separately) and confirm plant genotypes via DNA extraction and PCR. We also tested the expression and transcript levels of these proteins via RNA extraction and reverse transcriptase PCR, to ensure that the protein fluorescence intensity is solely dependent on degradation. We are in the process of confirming localization to the chloroplast via immunoblotting and confocal microscopy. Hopefully, our hypothesis that the N-end rule operates in chloroplasts will be confirmed by the assays and will lead to a greater understanding of protein dynamics, as well as uncover new avenues of biological research.
This summer, I’ve learned a wealth of new information and techniques! I had never done plant science research before interning at BTI, so I was able to explore a whole new facet of biology this summer. Through my work in the wet lab, I’ve gained a better understanding of how to plan and execute a long-term project. I’ve also learned how to perform several new experimental techniques including plant transformations, DNA and RNA extraction, immunoblotting, and confocal microscopy. Outside of the lab, the weekly seminars and the bioinformatics course we had the privilege of attending shed some light on numerous avenues of research under the plant science umbrella and exposed me to a whole new realm of biology. Additionally, through the various discussion panels we attended, I gained invaluable insight such as how to apply to graduate school programs, how to secure funding, and how to successfully network with a possible employer. Aside from making strides in both my academic and professional careers, I loved exploring Ithaca and the surrounding area during my free time!