ABSTRACT
The 26S proteasome is the major eukaryotic ATP-dependent protease, yet the detailed mechanisms used by the proteasomal heterohexameric AAA+ unfoldase to drive substrate degradation remain poorly understood. To perform systematic mutational analyses of individual ATPase subunits, we heterologously expressed the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro. Our studies demonstrate that the six ATPases have distinct roles in degradation, corresponding to their positions in the spiral staircases adopted by the AAA+ domains in the absence or presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. Whereas the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits three-fold symmetry with contributions from alternate subunits. These diverse functional asymmetries highlight how the 26S proteasome deviates from simpler, homomeric AAA+ proteases.
Subject(s)
Endopeptidase Clp/metabolism , Proteasome Endopeptidase Complex/metabolism , Endopeptidase Clp/chemistry , Escherichia coli/enzymology , Proteasome Endopeptidase Complex/chemistry , Protein Conformation , Saccharomyces cerevisiae/enzymology , Substrate SpecificityABSTRACT
The 26S proteasome is the major ATP-dependent protease in eukaryotes and thus involved in regulating a diverse array of vital cellular processes. Three subcomplexes form this massive degradation machine: the lid, the base, and the core. While assembly of base and core has been well-studied, the detailed molecular mechanisms involved in formation of the nine-subunit lid remain largely unknown. Here, we reveal that helices found at the C terminus of each lid subunit form a helical bundle that directs the ordered self-assembly of the lid subcomplex. Furthermore, we use an integrative modeling approach to gain critical insights into the bundle topology and provide an important structural framework for our biochemical data. We show that the helical bundle serves as a hub through which the last-added subunit Rpn12 monitors proper lid assembly before incorporation into the proteasome. Finally, we predict that the assembly of the COP9 signalosome depends on a similar helical bundle.
Subject(s)
Proteasome Endopeptidase Complex/chemistry , Protein Multimerization , Amino Acid Sequence , COP9 Signalosome Complex , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Peptide Hydrolases/chemistry , Protein Folding , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes polyubiquitinated substrates. Here we used electron microscopy and a new heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle from yeast. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes and the protein unfolding machinery at subnanometre resolution, outlining the substrate's path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.