Function and Assembly of Eukaryotic Proteasome

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DESCRIPTION (provided by applicant): Many complex molecular transactions in the cell are catalyzed by large multisubunit protein machines. Often, these machines are organized into ring-shaped structures, creating a central channel or chamber, and macromolecules are moved into or out of these chambers, usually by integral ATP-hydrolyzing (ATPase) domains or by noncovalently associated ATPase complexes, which may themselves have a toroidal organization. The 26S proteasome, the central intracellular protease of eukaryotic cells, is among the most intricate of such ring-based ATP- driven machines. It consists of a cylindrical core particle, the 20S proteasome, which houses a central proteolytic chamber, and a 19S regulatory particle (RP) on each end. The RP includes six different ATPase subunits, also likely to be in a ring-shaped subcomplex, which binds and unfolds protein substrates and drives them into the 20S proteasome proteolytic chamber. How such complicated ring-shaped complexes are assembled in vivo is poorly understood, and this is certainly true for the assembly of the 20S proteasome, which has four heteroheptameric rings. Even less clear is the assembly mechanism of the ~20-subunit RP. The long-range goal of this application is to delineate the pathway(s) of proteasome biogenesis in vivo. The proteasome has emerged as an important target for anti-cancer treatment and other therapies. Interfering with its assembly could provide a useful new approach for pharmaceutical intervention. The proposed experiments use a combination of genetic, biochemical, and biophysical methods and are centered on the model eukaryote Saccharomyces cerevisiae, which has a 26S proteasome very similar to the human complex. A major focus of the project will be on deciphering the pathway(s) by which the 20S proteasome assembles in vivo, including the identification of potential assembly factors (Specific Aim 1). Steps in 20S proteasome assembly will be reconstituted in vitro or in a bacterial co-expression system and the mechanisms of putative assembly factors will be tested (Specific Aim 2). A final set of experiments addresses the question of how the RP assembles, an issue for which there is only minimal information at present (Specific Aim 3). A potential RP assembly factor recently discovered in the PI's laboratory provides the starting point for these studies.