A Framework for Studying Crucial Steps in Proteasome Core Particle Assembly

Abstract

The ability of proteins to repeatedly and reliably self-assemble in the cell is a critical element of the maintenance of life. Despite this, the mechanisms that underlie these events are poorly understood. The proteasome core particle from Rhodococcus erythropolis is an excellent model system for understanding assembly processes. This system has a two step assembly pathway where individual subunits first assemble into half proteasomes. Then, two half proteasomes dimerize to produce a full proteasome core particle. The beta subunit of this complex is synthesized in an inactive with an N-terminal propeptide that is cleaved after assembly is complete, rendering the CP enzymatically active. Evidence suggests that the propeptides plays a crucial role in both steps of the assembly process. To date, however, it has been impossible to fully characterize the role of the propeptide in assembly because this protein is typically produced as a heterogeneous mixture with a variety of N-terminally truncations in the propeptides itself. Here, we used Ligation Independent Cloning to produce a beta variant, which we call D3, that is homogeneous for the full-length propeptide. We also used Native PAGE to begin to characterize the kinetics of half proteasome dimerization. We found that there is a temperature-dependent effect on the dimerization process and that the presence of the full propeptide dramatically slowed assembly when compared to the heterogeneous beta. Using these methods, we can now study the thermodynamics and kinetics of this system much more rigorously than has been possible to date.