Mentor 1

Matt Christians


Plants utilize a complex system of light responsive pathways to initiate discrete changes in the plant cell’s growth and development. The light regulating BTB (LRB) E3 ligase is utilized in the ubiquitinproteasome system (UPS) to target a group of photoreceptors, the phytochromes, for degradation. The UPS allows for the selective tagging and degradation of proteins in the cell. The phytochrome B complex is stable in FR, but broken down in red light by the LRBs. Evidence suggests that the LRBs become activated in red light by forming a complete E3 ligase complex which includes the protein Cul3. We propose to investigate how the LRBs become activated and bind to Cul3 in red light in the model plant Arabidopsis thaliana. Evidence suggests the LRBs are modified by the Nedd8 protein (i.e., a protein used to activate a small group of other proteins in eukaryotes) in response to red light. This project proposes to investigate whether the LRBs are modified by the Nedd8 protein by using an in vitro neddylation assay. The results of this assay will improve the understanding of how LRB E3 ligases function in modifying light responses in plants and will also provide insight into neddylation and its effect on protein activity.

To test our hypothesis that the LRB proteins are neddylated, in vitro testing will be used which limits the need for the genetic transformation of Arabidopsis to express the necessary tagged proteins needed for the assay. The assay will be performed using the Abcam Neddylation assay kit that includes Nedd8, along with other components necessary for neddylation with in vitro testing. We will test for neddylation using full-length LRB as well as C terminal and N terminal portions of LRB. For a positive control, CUL3a will be used as that is shown to be neddylated under standard in planta conditions. RBX1 may also be included in any neddylation assay since it has been found to increase neddylation rates. As a negative control, the C-terminus end of LRB1 will be assayed for neddylation as that domain is not hypothesized to be neddylated.

The results to date do not influence the hypothesis of whether neddylation occurs on the LRB proteins as any of the results obtained are in the preparation of the proteins needed to conduct the neddylation assay. Therefore, no direct findings as to the ability or inability of neddylation of LRBs have been found. The results of the protein production and preparation have been progressing, and the purification of the LRB-full length is upcoming providing the first substrate for the neddylation assay. This progress is in support of the future investigation of neddylation.

*This scholar and faculty mentor have requested that only an abstract be published.