Our long term goal is to obtain a better understanding of energy-dependent proteolysis in the cell, especially as it relates to the metabolically diverse Archaea which play a major role in global carbon mineralization and the production of greenhouse gases. Surprisingly, little is known about regulated protein turnover in the Archaea which are closely related to the Eucarya. Energy-dependent proteolysis is now known to be central to the regulation of cell division, metabolism, transcription, and other essential functions directly related to human health. Proteases which mediate energy-dependent hydrolysis comprise a small group of structurally related proteins. These proteases form a compartment with narrow openings which isolate the proteolytic active-sites away from the cytoplasmic constituents, thus, avoiding, non-specific protein degradation. An energy-dependent component of the protease is needed for recognition and/or unfolding of substrates which are then fed into the proteolytic chamber. This energy-dependence provides a proofreading step to insure that the proper substrate has been selected for destruction. Although energy-dependent proteases are predicted based on archaeal genome sequences, the only component which has been purified is the 20S proteasome. These 20S proteasomes only act on unfolded proteins in vitro and, thus, have not facilitated the identification of native substrates in the archaeal cell. The main objective of this study is to examine proteasome-mediated degradation in the archaeon Haloferax volcanii using genetic methods to determine in vivo proteasome functions. To achieve these goals, the 20S proteasome mRNA transcripts and protein levels will be analyzed under a variety of culture conditions which will provide information of the regulation, promoters, and operons of the proteasomes. The levels of active proteasome proteins will also be modified through chromosomal mutagenesis as well as the homologous express of epitope-tagged proteasome genes from plasmids. These approaches will enable us to link the in vivo level of the various proteasome proteins with cell phenotype(s) and, thus, provide an understanding of the role of these proteins in archaeal physiology. Furthermore, expression of the epitope-tagged proteasome subunits in H. volcanii will enable us to determine the arrangement of the alpha1, alpha2 and beta subunits in the 20S proteasomes. Several genetic methods are also presented which will facilitate the identification of foreign and native proteins which are degraded by the proteasome in the cell. This study will, therefore, unveil pathways which are regulated by the proteasomes and lay a foundation for understanding the biology of proteolysis as a mechanism of post-transcriptional regulation in this unusual group of organisms.
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