Proteolysis has been shown to have a prominent role in both cellular maintenance, and regulation of developmental and metabolic circuits in prokaryotes and eukaryotes. In particular, a specific enzyme complex, the 20S proteasome, has been identified as the primary component of eukaryotic protein degradation. Although this enzyme has been identified in both Bacteria and Archaea, role(s) for the 20S proteasome in these organisms remain unclear. However, the 20S proteasomes of Bacteria and Archaea provide both a simplified model system to investigate the function and activity of this enzyme complex, and a unique opportunity to establish relationships between common enzymes found throughout biology. This proposal addresses the function of the 20S proteasome in cell physiology of a methanoarchaeal species, Methanosarcina thermophila. A combination of genetic and biochemical techniques will be employed to elucidate potential physiological roles for this enzyme, and identify cellular factors which mediate the regulation and selectivity of this proteolytic enzyme. Using techniques for transformation of Methanosarcina species, strains lacking 20S proteasome activity will be constructed and examined for potential growth phenotypes. In addition, M. thermophila cellular components that interact with the 20S proteasome will be identified by characterizing higher order 20S proteasome-containing complexes on the basis of modified activity or size.
