Abstract by Michael Mann
Chemistry and Biochemistry
Role of the cytosolic chaperonin CCT in mTOR complex formation
Our work is focused on understanding the role of the cytosolic chaperonin containing tailless polypeptide TCP-1 (CCT) in mechanistic target of rapamycin (mTOR) complex assembly. Our lab has discovered that CCT binds to Raptor and mLST8, two subunits of the mTOR complexes. To investigate the effect CCT has on the assembly of these subunits into mTOR complexes, we are developing a method to rapidly remove CCT from cells using the insertion of an Auxin-Inducible Degron (AID) tag through CRISPR-targeted homologous recombination. Upon insertion of the AID tag on CCT, over-expression of OsTIR (an SCF E3 ubiquitin ligase in plants) and treatment with auxin will lead to rapid, specific, reversible degradation CCT. We have achieved a monoallelic knock-in of the AID-tag on CCTa, and have proposed alternative ways to achieve the complete knock-in. We will use AID-CCT containing cells to measure the effects of acute CCT loss on formation of mTOR complexes and folding of mLST8 and Raptor.