Structure Basis For The Activation Of 20S Proteasome By REG γ

Proteasomes are barrel-like structure, which is responsible for the degradation of large varieties of proteins in the nucleus and the cytoplasm. The axial pore of the proteasome is opened by the activators, so that the substrates can enter its catalytic chamber. Three calasses of activators have been known, including 19S(ortholog in archaea is called PAN), 11S(REGα,β,γ,ortholog in Tryponoma is called PA26), PA200(ortholog in yeast is called Blm10). The analysis of the structure of 19S, REGα, PA26, Blm10 or their complexes with 20S by crystallography or electron microscopy has been reported, nevertheless little information is known about the structure of REGy or REGy-20S complex. Therefore we are devoted to the study of the mechanism of the assembly of REGy-20S.The result of Co-IP and yeast two hybrid assay shows that REGy specifically associate with α7 subunit of 20S core particle.The key amino acid in the assembly of REGy-20S. such as P245,Y254 in REGγ,Y8, D9 in 20S, were found by mapping and site-directed mutagenesis Moreover, we proposed binding model of REGγ-20S. To validate this model,high concentration, high purity no-tagged REGy was purified.Subsequently,we will purify GST-20S from yeast and incubate it with REGy, in order to acquire the image of REGy-20S in CryoEM.