Mutational studies on differential proteasome activation by the 11S regulators

The eukaryotic 20S proteosome contains three distinct catalytic ? subunits that display chymotrypsin-like (CT), trypsin-like (T) and peptidylglutamyl preferring hydrolytic (PGPH) activities. Facing the proteasome's central chamber, the active sites are insulated from external solvent by N-terminal segments of the proteasome's ? subunits. For activation, a channel must be opened from the external solvent to the proteasome's central chamber. Two protein complexes bind the ends of the proteosome and activatie it, the 19S regulatory complex (RC) and the 11S regulator (11S REG). The 11S REG family is comprised of three subunits, ?, ? and ?. REG? and REG? active all three proteosome ? catalytic subunits whereas REG? stimulates only T-like activity. My dissertation aimed to determine the structural basis for differential proteosome activation by the REG subunits using site-directed and random mutagenesis. In the site directed mutagenesis study, I exchanged regions where the sequences diverge and characterized the resulting chimeras. Exchanged regions included the N-terminal, C-terminal and the activation loop flanking sequences. In the random mutagenesis study, REG? mutants bearing random mutations were screened for their ability to stimulate the proteasomal CT-like and PGPH activities. I found that none of the divergent regions controls the activation of specific proteosome ? subunits. Rather, substitution of REG?Lys188 broadens proteosome activation of that homolog to match the activation by REGs ? and ?. My studies also provide strong biochemical evidence for all allosteric basis for differential proteosome activation by REG subunits. I conclude that the restricted proteosome activation by REG is not due to the selective entry or exit of substrates to the proteosome through REG? rings, but is due to large conformational adjustments in the proteosome upon binding the more ridge REG? heptamer. I hypothesize that substitution of Lys188 by Glu or Asp increases the plasticity of REG? heptamers and relieves the proteasomal adjustments. Thereby allowing all three catalytic subunits to remain active upon channel opening by REG?