Interaction of plant seryl-tRNA synthetase and BEN1, protein involved in brassinosteroid metabolism

Aminoacyl-tRNA synthetases (AARS) are essential cellular enzymes that play significant role in protein synthesis by charging amino acids to their cognate tRNAs. In recent years more evidence appeared showing involvement of these enzymes in diverse cellular functions beyond translation. Characterization of these non-canonical functions broadens our knowledge in functional proteomics. The studies of aaRS assemblies in plants are scarce. Our main research goals were to determine potential protein interacting partners of seryl- tRNA synthetase (SerRS) in plant cell, its potential nuclear localization and kinetic characterization. In search for SerRS interacting protein partners in plant Arabidopsis thaliana, L. we conducted yeast-two hybrid (Y2H) screen on cDNA libraries and tandem affinity purification (TAP) in plant. Potential interacting partners were identified either by DNA sequencing (Y2H) or mass spectrometry (TAP). BEN1, protein involved in metabolism of brassinosteroid hormones, was identified as the most promising interacting partner. Interaction of BEN1 and SerRS was analyzed in vitro using isothermal calorimetry titration (ITC), pull-down and surface plasmon resonance method (SPR). Probably due to the nature of interaction we were not able to retrieve positive results using pull-down assay and ITC, but SPR gave us positive confirmation and information about dissociation constant. To determine regions responsible for protein-protein interaction we planned to prepare shortened variants of both SerRS and BEN1 proteins and use them in biophysical analysis. So far we managed to construct, successfully express and purify BEN1 variant lacking 37 amino acids long polar region at the N-terminus and SerRS variant lacking hydrophilic region at C-terminus. Although we successfully cloned other truncated variants of SerRS protein, thus far their expression yielded inclusion bodies. Biophysical determination of possible interactions between SerRS and BEN1 variants will give us insight in additional cell functions and physiology of both SerRS and BEN1 proteins.