Megan Johnston

Project Summary:

The CLP protease system is conserved in bacteria, mitochondria, and higher plant plastids, such as chloroplasts, and functions to maintain protein homeostasis by degrading a wide variety of substrates. Several CLP protease components have been identified in chloroplasts; however, more components are believed to be involved in regulating this complex degradation system. As such, affinity purification and mass spectrometry experiments were conducted by the van Wijk lab to identify interactors with the CLP protease system. The proteins UVR2 and UVR3 were found in a stabilized CLPC1 chaperone-protease complex, distinguishing UVR2 and UVR3 as candidates for regulating CLP proteolysis. These proteins are especially of interest as UVR2 and UVR3 contain UVR motifs, which are also found in three proteins already identified to be involved in the chloroplast CLP protease system: CLPC1, CLPC2, and CLPF. In bacteria, adaptors regulate assembly/activation of the chaperone proteins CLPB and CLPC through direct interactions with their UVR motifs. While these proteins contain a UVR motif, UVR2 and UVR3 have no previously characterized function. As such, this study aims to understand the role that UVR2 and UVR3 play in the chloroplast CLP protease system. My specific aims were to test whether UVR2 and UVR3 form dimers and interact with CLP proteins in vitro, as well as determine if these proteins serve a redundant function in stress response. To test UVR2 and UVR3 interactions, I generated recombinant UVR2 and UVR3 proteins that contain either a GST-tag or a His-tag using restriction cloning for future in vitro interaction testing. UVR2 and UVR3 have an almost (81%) identical protein sequence. To determine if these proteins serve a redundant function and if that function is involved in stress response, I established a double null uvr2uvr3 t-DNA insertion line, which can be used for stress response phenotype studies in the future. My objective was to characterize the function of UVR2 and UVR3 and how these proteins interact with the CLP protease system, as unraveling the mechanisms of substrate selection by the CLP protease system is key to understanding chloroplast proteostasis.


My Experience:

The Boyce Thompson Institute Summer REU Internship Program will be an experience that I will never forget. I was able to pursue my passion for plants, getting the opportunity to expand my knowledge of plant science research in the van Wijk lab. The people in my lab were so welcoming and made such a fun learning environment! My research mentor, Marissa, was amazing! She took the time to make sure I fully understood the techniques I was going to be using. Her support this summer allowed me to build independence and confidence when conducting research. Outside of research, I made wonderful memories with friends and got to explore the nature that Ithaca has to offer.