CAND1 STRUCTURE-FUNCTION

The first part of my thesis research is focused on the role of Cullin-Associated NEDD8 Dissociated Protein 1 (CAND1) in Arabidopsis reproductive organ development. CAND1 is one of the key regulators of the SKP1-CUL1/RBX1-F-BOX (SCF) complex. Akin to an exchange factor, CAND1 functions by increasing the Koff of F-box proteins and facilitating the recycling of the limited Cullin-scaffold. The importance of CAND1 in recycling the Cullin-scaffold has led to the synthesis of adaptive exchange hypothesis. Adaptive exchange hypothesizes that cells lacking CAND1 are not adapted well to handle larger pool of F-BOX proteins. In support of this hypothesis, a recent study illustrated that CAND-deficient cells died upon F-BOX overexpression. However, the wild type cells remained viable. In view of this, the first portion of my thesis was focused on testing the adaptive exchange hypothesis. Our study found that there was a pollen death in Arabidopsis cand1-3 homozygous mutant. In addition, bioinformatics analysis showed that there is a relatively higher F-BOX protein transcript pool compared to all the tissues analyzed. These results are consistent with the notion of the adaptive exchange hypothesis.

The second portion of my thesis research is focused on understanding the dynamics of Arabidopsis SCF E3 ligase protein complex. Dynamics of protein complexes could be studied by performing protein-protein interactions (PPI) assays. However, no study till date has expressed and purified Arabidopsis SCF E3 ligase core proteins to perform PPI assays. Therefore, my aim for the second portion of my thesis was to express, purify, and obtain the structure of Arabidopsis SCF core proteins. As such, I was able to successfully express, and purify Arabidopsis CAND1-CUL1/RBX1 protein complex.