PROPERTIES OF AN ATP/UBIQUITIN-DEPENDENT PROTEASE

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The 26S proteasome is an extremely large (2,000,000 Da) multisubunit enzyme that degrades important regulatory proteins such as p53, cyclins, NfkappaB, Sic1, etc. It is comprised of a spherical regulatory complex (RC) containing 18 different subunits that associates with one or both ends of the cylindrical 20S proteasome. Over the past six years, we cloned and expressed 9 of the RC subunits. Several other groups identified the remaining subunits, and today we know the sequences of all 18 subunits. Although the crystal structure of the 20S proteasome has been solved, the arrangement of RC subunits is largely unknown. Because it is important to position the RC subunits relative to one another, we have used Far Western blotting and in vitro assembly reactions to determine intersubunit contacts among 9 of the subunits. We will continue these studies adding limited dissociation of the RC in urea and mass spectrometry to analyze the arrangement of subunits in the regulatory complex. Another important goal is to assign functions to the RC subunits. Six RC subunits belong to a family of ATPases approximately 400 amino acids in length. Although the central regions in the ATPases are highly conserved, the N-terminal 150 residues and C-terminal sequences are divergent. Having constructed a series of chimeric ATPases we will determine whether the N-terminal and/or C- terminal regions confer distinct functions to the ATPases. Several years ago we found that one RC subunit (5a) binds polyubiquitin chains. We recently discovered that a dimer of subunits S2 and S4 also binds polyUb chains. We will characterize the polyUb binding site(s) within these proteins. Finally, we have found that four RC subunits can be crosslinked to a peptide corresponding to a C-terminal extension of a 20S proteasome subunit. We will examine the peptide binding properties of RC subunits because some RC components may bind proteasome extensions to assemble the 26S proteasome and others may bind unfolded regions in proteolytic substrates. The 26S proteaseome is clearly involved in control of the cell cycle, and it likely generates antigenic peptides that are displayed on MHC Class I molecules. For these reasons, increased knowledge of the 26S proteasome should have significant medical implications.