Differential Roles of the COOH Termini of AAA Subunits of PA700 (19 S Regulator) in Asymmetric Assembly and Activation of the 26 S Proteasome (original) (raw)
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Assembly of the regulatory complex of the 26S proteasome
Molecular biology reports, 1999
The 19S regulatory complex (RC) of 26S proteasomes is a 900-1000 kDa particle composed of 18 distinct subunits (S1-S15) ranging in molecular mass from 25 to 110 kDa. This particle confers ATP-dependence and polyubiquitin (polyUb) recognition to the 26S proteasome. The symmetry and homogenous structure of the proteasome contrasts sharply with the remarkable complexity of the RC. Despite the fact that the primary sequences of all the subunits are now known, insight has been gained into the function of only eight subunits. The six ATPases within the RC constitute a subfamily (S4-like ATPases) within the AAA superfamily and we have shown that they form specific pairs in vitro. We have now determined that putative coiled-coils within the variable N-terminal regions of these proteins are likely to function as recognition elements that direct the proper placement of the ATPases within the RC. We have also begun mapping putative interactions between non-ATPase subunits and S4-like ATPases. ...
Journal of Biological Chemistry, 2021
The proteasome is a large protease complex that degrades many different cellular proteins. In eukaryotes, the 26S proteasome contains six different subunits of the ATPases associated with diverse cellular activities family, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single Rpt-like ATPases associated with diverse cellular activities ATPase, the proteasome-activating nucleotidase, which forms a trimer of dimers. A key proteasomeactivating nucleotidase proline residue (P91) forms cisand transpeptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. However, the importance of the equivalent Rpt prolines for eukaryotic proteasome assembly was unknown. Here we showed that the equivalent proline is highly conserved in Rpt2, Rpt3, and Rpt5, and loosely conserved in Rpt1, in deeply divergent eukaryotes. Although in no case was a single Pro-to-Ala substitution in budding yeast strongly deleterious to growth, the rpt5-P76A mutation decreased levels of the protein and induced a mild proteasome assembly defect. Moreover, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all caused synthetic defects when combined with deletions of specific proteasome base assembly chaperones. The rpt2-P103A rpt5-P76A double mutant had uniquely strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant formed aggregates that were cleared, at least in part, by Hsp42 chaperonemediated protein quality control. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.
Insights into the molecular architecture of the 26S proteasome
Proceedings of the National Academy of Sciences, 2009
Cryo-electron microscopy in conjunction with advanced image analysis was used to analyze the structure of the 26S proteasome and to elucidate its variable features. We have been able to outline the boundaries of the ATPase module in the ''base'' part of the regulatory complex that can vary in its position and orientation relative to the 20S core particle. This variation is consistent with the ''wobbling'' model that was previously proposed to explain the role of the regulatory complex in opening the gate in the ␣-rings of the core particle. In addition, a variable mass near the mouth of the ATPase ring has been identified as Rpn10, a multiubiquitin receptor, by correlating the electron microscopy data with quantitative mass spectrometry.
Regulatory subunit interactions of the 26S proteasome, a complex problem
Trends in Biochemical Sciences, 2000
The 26S proteasome is the major non-lysosomal protease in eukaryotic cells. This multimeric enzyme is the integral component of the ubiquitinmediated substrate degradation pathway. It consists of two subcomplexes, the 20S proteasome, which forms the proteolytic core, and the 19S regulator (or PA700), which confers ATP dependency and ubiquitinated substrate specificity on the enzyme. Recent biochemical and genetic studies have revealed many of the interactions between the 17 regulatory subunits, yielding an approximation of the 19S complex topology. Inspection of interactions of regulatory subunits with non-subunit proteins reveals patterns that suggest these interactions play a role in 26S proteasome regulation and localization.
The central unit within the 19S regulatory particle of the proteasome
Nature Structural & Molecular Biology, 2008
The 26S proteasome is a multisubunit enzyme composed of a cylindrical catalytic core (20S) and a regulatory particle (19S) that together perform the essential degradation of cellular proteins tagged by ubiquitin. To date, however, substrate trajectory within the complex remains elusive. Here we describe a previously unknown functional unit within the 19S, comprising two subunits, Rpn1 and Rpn2. These toroids physically link the site of substrate recruitment with the site of proteolysis. Rpn2 interfaces with the 20S, whereas Rpn1 sits atop Rpn2, serving as a docking site for a substrate-recruitment factor. The 19S ATPases encircle the Rpn1-Rpn2 stack, covering the remainder of the 20S surface. Both Rpn1-Rpn2 and the ATPases are required for substrate translocation and gating of the proteolytic channel. Similar pairing of units is found in unfoldases and nuclear transporters, exposing common features of these protein nanomachines.
Assembly, structure, and function of the 26S proteasome
Trends in cell biology, 2010
The 26S proteasome is a large multi-protein complex involved in the regulated degradation of ubiquitinated proteins in the cell. The 26S proteasome has been shown to control an increasing number of essential biochemical mechanisms of the cellular lifecycle including DNA synthesis, repair, transcription, translation and cell signal transduction. Concurrently, it is increasingly seen that malfunction of the ubiquitin proteasome system contributes to the pathogenesis of disease. The recent identification of four molecular chaperones, in addition to five previously identified chaperones, have provided mechanistic insight into how this cellular megastructure is assembled in the cell. These data, together with new insights into the structure and function of the proteasome, provide a much better understanding of this complex protease.
Reconfiguration of the proteasome during chaperone-mediated assembly
Nature, 2013
The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric a-ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt carboxy-terminal tails inserting into pockets of the a-ring 1-4 . Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit 5-10 . Here we report that the base subassembly of the Saccharomyces cerevisiae proteasome, which includes the Rpt ring, forms a high-affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6 and Rpn14. Chaperone-mediated dissociation was abrogated by a non-hydrolysable ATP analogue, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound a-pockets with poor specificity, except for Rpt6, which uniquely bound the a2/a3-pocket. Although the Rpt6 tail is not visualized within an a-pocket in mature proteasomes 2-4 , it inserts into the a2/a3-pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.
Toward an integrated structural model of the 26S proteasome
Molecular & cellular proteomics : MCP, 2010
The 26S proteasome is the end point of the ubiquitinproteasome pathway and degrades ubiquitylated substrates. It is composed of the 20S core particle (CP), where degradation occurs, and the 19S regulatory particle (RP), which ensures substrate specificity of degradation. Whereas the CP is resolved to atomic resolution, the architecture of the RP is largely unknown. We provide a comprehensive analysis of the current structural knowledge on the RP, including structures of the RP subunits, physical protein-protein interactions, and cryoelectron microscopy data. These data allowed us to compute an atomic model for the CP-AAA-ATPase subcomplex. In addition to this atomic model, further subunits can be mapped approximately, which lets us hypothesize on the substrate path during its degradation.
Multiple Associated Proteins Regulate Proteasome Structure and Function
Molecular Cell, 2002
We have identified proteins that are abundant in affinity-purified proteasomes, but absent from proteasomes as previously defined because elevated salt concentrations dissociate them during purification. The major components are a deubiquitinating enzyme (Ubp6), a ubiquitin-ligase (Hul5), and an uncharacterized protein (Ecm29). Ecm29 tethers the proteasome core particle to the regulatory particle. Proteasome binding activates Ubp6 300-fold and is mediated by the ubiquitin-like domain of Ubp6, which is required for function in vivo. Ubp6 recognizes the proteasome base and its subunit Rpn1, suggesting that proteasome binding positions Ubp6 proximally to the substrate translocation channel. ubp6Δ mutants exhibit accelerated turnover of ubiquitin, indicating that deubiquitination events catalyzed by Ubp6 prevent translocation of ubiquitin into the proteolytic core particle.