The 26S proteasome is at the executive end of the ubiquitinproteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The "lid" of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAAATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.coiled coils | mass spectrometry | proteasome-COP9-eIF3 domain | proteasome-cyclosome repeats I n eukaryotes, the ubiquitin-proteasome pathway (UPP) is essential for proteostasis: Misfolded proteins or otherwise defective proteins as well as short-lived regulatory proteins are eliminated by degradation (1). The UPP regulates many fundamental cellular processes, such as protein quality control, DNA repair, and signal transduction (for review see ref.2). The 26S proteasome is the most downstream element of the UPP, executing the degradation of polyubiquitylated substrates (3-5). It consists of the barrelshaped core particle (CP; approximately 700 kDa), which sequesters the proteolytically active site in its central cavity, and the regulatory particle (RP; approximately 900 kDa), which is attached at either one or both ends of the CP and prepares substrates for degradation (6).The RP consists of 19 different canonical subunits, including six regulatory particle AAA-ATPase subunits (Rpt1-6) and 13 regulatory particle non-ATPase subunits (Rpn1-3, Rpn5-13, and Rpn15). The integral ubiquitin (Ub) receptors Rpn10 and Rpn13 recognize polyubiquitylated substrates (7-9). Alternatively, polyubiquitylated substrates can be recruited by shuttling Ub-receptors (Dsk2, Rad23, Ddi2), which bind to substrates with their Ub-associated domain, and to Rpn1, Rpn10, or Rpn13 at their Ub-like domain (5). The metalloprotease Rpn11 deubiquitylates substrates prior to their degradation (10, 11). The functions of the other Rpn subunits remain to be established. The AAA-ATPases form a hexameric ring that unfolds substrates, opens the gate to the CP, and eventually translocates the substrates to the CP.Electron microscopy (EM) (12) and X-...

The 26S proteasome operates at the executive end of the ubiquitinproteasome pathway. Here, we present a cryo-EM structure of the Saccharomyces cerevisiae 26S proteasome at a resolution of 7.4 Å or 6.7 Å (Fourier-Shell Correlation of 0.5 or 0.3, respectively). We used this map in conjunction with molecular dynamics-based flexible fitting to build a near-atomic resolution model of the holocomplex. The quality of the map allowed us to assign α-helices, the predominant secondary structure element of the regulatory particle subunits, throughout the entire map. We were able to determine the architecture of the Rpn8/Rpn11 heterodimer, which had hitherto remained elusive. The MPN domain of Rpn11 is positioned directly above the AAA-ATPase N-ring suggesting that Rpn11 deubiquitylates substrates immediately following commitment and prior to their unfolding by the AAA-ATPase module. The MPN domain of Rpn11 dimerizes with that of Rpn8 and the C-termini of both subunits form long helices, which are integral parts of a coiled-coil module. Together with the C-terminal helices of the six PCI-domain subunits they form a very large coiled-coil bundle, which appears to serve as a flexible anchoring device for all the lid subunits.protein degradation | electron microscopy | deubiquitylating enzyme T he 26S proteasome is a 2.5 MDa molecular machine designed for the controlled degradation of proteins marked for destruction by the covalent attachment of polyubiquitin chains [for reviews see (1-3)]. It is composed of two copies, each of 33 canonical subunits, as well as some proteasome interacting proteins (PIPs). The 26S holocomplex comprises two types of subcomplexes: the cylindrical 20S core particle (CP) harbouring the proteolytic chamber and the two 19S regulatory particles (RPs), which attach to opposite ends of CP cylinder. The RPs have multiple roles in preparing substrates for degradation: They recognize and bind ubiquitylated proteins, they deubiquitylate them followed by their unfolding, and they control the opening of the gate which gives access to the interior of the CP.While the structure of the 20S core complex was determined by X-ray crystallography almost two decades ago (4, 5), the structure of the 26S complex remained recalcitrant to crystallization attempts, presumably due to its conformational and compositional heterogeneity. Recently, the subunit architecture of the holocomplex has been determined by cryo-electron microscopy (EM) single particle analysis (SPA and ref. 6, 7) independently by two groups using different approaches for the assignment of RP subunits. Lander, et al. (6) obtained a 9 Å resolution map (Fouriershell correlation, FSC ¼ 0.5) of the 26S Saccharomyces cerevisiae proteasome and they determined the subunit positions by means of fusion constructs and automated segmentation methods. Lasker, et al. (7) performed an exhaustive computational search of possible subunit configurations within the boundaries of an 8.5 Å map of the 26S proteasome from Schizosaccharomyces pombe scoring possible configurati...

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Summary Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here we address the elusive link between these phenomena employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes.

The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATP h ) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATP h conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle.conformational switching | proteolysis | proteostasis | quality control I n the ubiquitin-proteasome pathway (UPP) the 26S proteasome performs the degradation of intracellular proteins marked for destruction by the covalent attachment of polyubiquitin chains (1-5). The 2.5 MDa complex consists of the barrel-shaped 20S core particle (CP) as well as one or two copies of the 19S regulatory particle (RP) controlling the entry of substrates into the proteolytic chamber of the CP. The structure of the CP was solved by X-ray crystallography a long time ago (6, 7); whereas, the molecular architecture of the 26S holocomplex was determined only recently using cryo-EM single-particle analysis (SPA) approaches (8-10). The RP comprises a ringshaped AAA-ATPase heterohexamer (Rpt1-6) responsible for substrate unfolding and translocation across the CP gate and 13 RP non-ATPases (Rpn1-3, 5-13, 15) surrounding the AAAATPase module. The role of the Rpns is the acceptance of substrates and their deubiquitylation. For a full mechanistic understanding of the early steps of substrate processing it is essential to reveal its dynamics.The compositional and conformational heterogeneity of 26S proteasome preparations makes the structural characterization of this molecular machine challenging (11). Compositional heterogeneity results from multiple proteins that interact with the 26S proteasome substoichiometrically, such as deubiquitylating enzymes (DUBs) or shuttling ubiquitin (Ub) receptors. Conformational switching of the 26S proteasome is mostly driven by ATP binding and hydrolysis. Each of the six distinct Rpt subunits is able to bind and hydrolyze ATP (12-14), which may...

In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA + ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA + ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.26S proteasome | cryo-electron microscopy | AAA + ATPase | integrative modeling | single-particle analysis I n eukaryotic cells, the ubiquitin-proteasome system (UPS) degrades proteins that are misfolded, damaged, or no longer needed (1). The 26S proteasome is a 2.5-MDa multisubunit complex comprising the barrel-shaped 20S core particle (CP), where degradation takes place, and one or two 19S regulatory particles (RPs), which bind to the ends of the CP (2-4). The CP is built of four coaxially stacked heteroheptameric rings of α-and β-subunits in the order of αββα (5). Three of the seven β-subunits are catalytically active; substrates are sequestered from the cellular environment in a chamber formed by the two β-rings (6, 7). This self-compartmentalization is a hallmark of many intracellular proteases (8). Substrate access to the proteolytic chamber is controlled by the α-subunit N-terminal extensions, forming a gate (3). Most of proteasome activators, including the RP, contain C-terminal hydrophobic-tyrosine-X (HbYX) motifs, which have been reported to insert into α-ring pockets, triggering gate opening (9-11).The RP is composed of at least 19 canonical subunits and interacts substoichiometrically with an array of proteasomeinteracting proteins that modulate RP function (3). The RP is divided into the "base" and the "lid" subcomplexes. The core of the base is formed by a heterohexameric ATPase associated with various cellular activities (AAA + ATPase), which is the driver of large-scale conformational dynamics of the RP. The AAA + ATPase prepares substrates for degradation in coordination with at least three ubiquitin receptors [26S proteasome non-ATPase regulatory subunit 1 (Rpn1), Rpn10, and Rpn13] (12-14) and a deubiquitylating subunit (Rpn11) (15, 16). Other subunits have structural roles, such as holding the CP and RP together, or in coordinating the movements needed to position the substrates above the pore of the AAA + ATPase for unfolding and translocation (17, 18). The AAA + ATPase is lined by aromatic-hydrophobic loops (pore-1 loops), wh...

The 26S proteasome is a key player in eukaryotic protein quality control and in the regulation of numerous cellular processes. Here, we describe quantitative in situ structural studies of this highly dynamic molecular machine in intact hippocampal neurons. We used electron cryotomography with the Volta phase plate, which allowed high fidelity and nanometer precision localization of 26S proteasomes. We undertook a molecular census of single- and double-capped proteasomes and assessed the conformational states of individual complexes. Under the conditions of the experiment—that is, in the absence of proteotoxic stress—only 20% of the 26S proteasomes were engaged in substrate processing. The remainder was in the substrate-accepting ground state. These findings suggest that in the absence of stress, the capacity of the proteasome system is not fully used.

SUMMARY The proteasome is the central protease for intracellular protein breakdown. Coordinated binding and hydrolysis of ATP by the six proteasomal ATPase subunits induces conformational changes that drive the unfolding and translocation of substrates into the proteolytic 20S core particle for degradation. Here, we combine genetic and biochemical approaches with cryo-electron microscopy and integrative modeling to dissect the relationship between individual nucleotide binding events and proteasome conformational dynamics. We demonstrate unique impacts of ATP binding by individual ATPases on the proteasome conformational distribution and report two conformational states of the proteasome suggestive of a rotary ATP hydrolysis mechanism. These structures, coupled with functional analyses, reveal key roles for the ATPases Rpt1 and Rpt6 in gating substrate entry into the core particle. This deepened knowledge of proteasome conformational dynamics reveals key elements of intersubunit communication within the proteasome and clarifies the regulation of substrate entry into the proteolytic chamber.