Docking of the Proteasomal ATPases' Carboxyl Termini in the 20S Proteasome's α Ring Opens the Gate for Substrate Entry

428

513

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-...

Section: Aaa-atpase Hexamermentioning

confidence: 53%

Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

Lasker

Förster

Bohn

et al. 2012

428

513

“…Three of the six AAA-ATPases (Rpt2, Rpt3, and Rpt5) share a C-terminal motif, a hydrophobic residue (Hb), and a tyrosine followed by a residue of any type (HbYX), which can bind to pockets in the CP (30). In the EM map, the C termini of Rpt3 and Rpt5 are clearly resolved, making it possible to discern bulky side chains (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structure of the human 26S proteasome at a resolution of 3.9 Å

Schweitzer

Aufderheide

Rudack

et al. 2016

187

220

Protein degradation in eukaryotic cells is performed by the UbiquitinProteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing.proteostasis | cryo-electron microscopy | AAA-ATPase | integrative modeling T he 26S proteasome is an ATP-dependent multisubunit protease degrading polyubiquitylated proteins (1, 2). It operates at the executive end of the Ubiquitin-Proteasome System (UPS) and has a key role in cellular proteostasis. The 26S proteasome selectively removes misfolded proteins or proteins no longer needed and it is critically involved in numerous cellular processes such as protein quality control, regulation of metabolism, cell cycle control, or antigen presentation. Malfunctions of the UPS are associated with various pathologies, including neurodegenerative diseases and cancer. Therefore, the proteasome is an important pharmaceutical target, and a high-resolution structure is a prerequisite for structure-based drug design (3).The 26S proteasome comprises the 20S cylindrical core particle (CP), where proteolysis takes place, and 19S regulatory particles (RPs). In cellular environments, 26S holocomplexes with either one or two RPs bound to the ends of the cylindershaped CP coexist (4). The role of the RPs is to recruit ubiquitylated substrates, to cleave off their polyubiquitin tags, and to unfold and translocate them into the CP for degradation into short peptides. Whereas X-ray crystallography has revealed the atomic structures first of archaeal 20S proteasome (5) and subsequently of the yeast (6) and mammalian proteasome (7), only lower-resolution structures were available for the 26S holocomplex. Given the compositional and conformational heterogeneity of the RP, single-particle cryo-electron microscopy (cryo-EM) has been the most successful approach to determining the structure of the 26S holocomplex (8). At this point, the most detailed insights have been obtained ...

“…The challenge will be to identify substrates degraded by one or more of these Cdc48-dependent mechanisms. Docking of C-terminal HbYX tripeptides into pockets on the periphery of the 20S α ring are important for 20S binding by archaeal PAN and the Rpt 1-6 unfolding ring of the 19S eukaryotic regulatory particle (10,19,21). HbYX-α interactions also help stabilize binding of Cdc48 to 20S, but archaeal Cdc48 still binds and functionally collaborates with 20S after deletion of this C-terminal tripeptide (4).…”

Section: Discussionmentioning

confidence: 99%

Bipartite determinants mediate an evolutionarily conserved interaction between Cdc48 and the 20 S peptidase

Barthelme

Sauer

2013

Proteasomes are essential and ubiquitous ATP-dependent proteases that function in eukarya, archaea, and some bacteria. These destructive but critically important proteolytic machines use a 20S core peptidase and a hexameric ATPase associated with a variety of cellular activities (AAA+) unfolding ring that unfolds and spools substrates into the peptidase chamber. In archaea, 20S can function with the AAA+ Cdc48 or proteasome-activating nucleotidase (PAN) unfoldases. Both interactions are stabilized by C-terminal tripeptides in AAA+ subunits that dock into pockets on the 20S periphery. Here, we provide evidence that archaeal Cdc48 also uses a distinct set of near-axial interactions to bind 20S and propose that similar dual determinants mediate PAN-20S interactions and Rpt 1-6 -20S interactions in the 26S proteasome. Current dogma holds that the Rpt 1-6 unfolding ring of the 19S regulatory particle is the only AAA+ partner of eukaryotic 20S. By contrast, we show that mammalian Cdc48, a key player in cell-cycle regulation, membrane fusion, and endoplasmic-reticulum-associated degradation, activates mammalian 20S and find that a mouse Cdc48 variant supports protein degradation in combination with 20S. Our results suggest that eukaryotic Cdc48 orthologs function directly with 20S to maintain intracellular protein quality control. AAA+ protease | p97A AA+ (ATPase associated with a variety of cellular activities) enzymes use cycles of ATP binding and hydrolysis to exert mechanical forces on substrates and power a diverse array of biological functions (1). In all cells, protein-unfolding machines of the AAA+ family form hexameric rings that cooperate with associated peptidases to execute intracellular protein degradation (2). The proteolytic sites of AAA+ proteases are sequestered within the interior of a barrel-shaped, self-compartmentalized peptidase, accessible only through narrow axial entrance tunnels that exclude folded proteins. Peptidase access is controlled by the AAA+ unfoldase, which docks with the peptidase, pulls the native structure of target proteins apart, and then translocates the polypeptide through its axial channel and into the degradation chamber.The 20S peptidase is the degradation module for proteasomes in all domains of life, but functions with different AAA+ unfoldases, including the Rpt 1-6 unfolding ring of the eukaryotic 26S proteasome, the double-ring Cdc48 or single-ring proteasomeactivating nucleotidase (PAN) enzymes in archaeal proteasomes, and the Mpa unfoldase in Actinobacteria (Fig. 1A) (3-6). In all organisms, the 20S peptidase has a four-ring α 7 β 7 β 7 α 7 structure and uses N-terminal residues of the α subunits to gate entrance of substrates into its degradation chamber (Fig. 1B) (5, 7-9).The subunits of the AAA+ ring have C-terminal tripeptides that dock into conserved pockets on the peptidase α ring and help stabilize an open-gate conformation to allow 20S degradation of proteins or large peptides (Fig. 1B) (10). These tripeptides generally match a hydrophobic, tyrosine, any resi...