Structure of the 26S proteasome from Schizosaccharomyces pombe at subnanometer resolution

Abstract

The structure of the 26S proteasome from Schizosaccharomyces pombe has been determined to a resolution of 9.1 Å by cryoelectron microscopy and single particle analysis. In addition, chemical cross-linking in conjunction with mass spectrometry has been used to identify numerous residue pairs in close proximity to each other, providing an array of spatial restraints. Taken together these data clarify the topology of the AAA-ATPase module in the 19S regulatory particle and its spatial relationship to the α-ring of the 20S core particle. Image classification and variance analysis reveal a belt of high "activity" surrounding the AAA-ATPase module which is tentatively assigned to the reversible association of proteasome interacting proteins and the conformational heterogeneity among the particles. An integrated model is presented which sheds light on the early steps of protein degradation by the 26S complex.deubiquitylating enzymes | macromolecular complex | ubiquitinproteasome pathway | ubiquitin receptor | single particle classification I n eukaryotic cells, most proteins in the cytosol and the nucleus are regulated via the ubiquitin-proteasome pathway and malfunctions of this pathway have been implicated in a wide variety of diseases (1). The 26S proteasome is the most downstream element of this pathway, executing protein degradation (2-4). Unlike constitutively active proteases, the proteasome has the capacity to degrade almost any protein, yet it acts with exquisite specificity. The key stratagem is self-compartmentalization: The active sites of the proteolytic 20S core particles (CPs) are sequestered from the cellular environment in the interior of this barrelshaped subcomplex (5). Proteins destined for degradation are marked by a polyubiquitin chain, a degradation signal that is recognized by the 19S regulatory particles (RPs) that bind to either one or both ends of the CP to form the 26S holocomplex. The RPs (i) recognize the polyubiquitylated substrates, (ii) trim and recycle the polyubiquitin chains, (iii) unfold substrates to be degraded, and (iv) open the gate to the CP and assist in substrate translocation into the interior of the CP. These tasks are performed by a complex machinery involving at least 19 different subunits, 6 AAA-ATPases (Rpt1-6), and 13 non-ATPases (Rpn1-3, Rpn5-13, Rpn15/Sem1p).Although the structure of the CP has been elucidated in great detail by X-ray crystallography (6, 7), the structure of the RP is only dimly understood at present. Best characterized are the AAA-ATPases which form a heterohexameric subcomplex situated at the base of the RP in close proximity to the α-rings of the CP (8, 9). The C-terminal residues of Rpt2 and Rpt5 were shown to be involved in opening the gate in the α-rings, allowing substrates to enter the CP. A similar mechanism has been postulated for proteasome-activating nucleotidase (PAN), the archaeal homohexameric homolog of the eukaryotic AAA-ATPase module (10). Crystal structures of the two major fragments of PAN suggest that the N-and C-terminal domains...

Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

“…For example, the AAA-ATP ring module of the S19 regulatory fraction of the proteasome (26) shows similar circumstances with the availability of an overall cryo-EM density map and reliable homology models for each subunit in the ring. Yet ambiguity exists in the ring arrangement and its registration to the S20 subunit.…”

“…At the same time, the complexity and size of the investigated protein complexes increased (23,24), with recent studies probing the 600 kDa PolIIF transcription system (25) and the intact proteasome (26). The combination of the technique with particle reconstruction by cryo-EM appears to be especially powerful because the cross-link data is helpful in assigning specific subunits to unassigned electron density.…”

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

“…The topological order of the AAA-ATPase subunits is Rpt1/Rpt2/ Rpt6/Rpt3/Rpt4/Rpt5, as determined based on protein-protein interactions (20) and engineered disulfide cross-links (21). Integration of a cryoelectron microscopy (cryo-EM) map at 9.1-Å resolution, protein-protein interactions, and site-specific intermolecular cross-links revealed the relative positions of the AAAATPase ring and the CP (20,22), which was subsequently corroborated by engineered disulfide cross-links between the AAAATPase C termini and residues in the pockets between the α-subunits of the CP (23). In contrast, the spatial configuration of the non-ATPase subunits and their role in the preparation of substrates for degradation remains largely unknown.…”

“…1A). Notably, we have approximately doubled the number of particles previously used for a single-particle reconstruction (22), to 375,000. This increase allowed us to improve the resolution from 9.1 to 8.4 Å (Fig.…”

High-Throughput Single Particle Cryo-Electron Microscopy Applied to the 26S Proteasome

“…Typically tens of thousands or even hundreds of thousands high-resolution particle images of consistent quality are required for classification and to calculate three-dimensional electron density maps. In recent years we have gradually refined [2,3] the structure of the 26S proteasome to a current resolution of approximate 9 Å (at 0.5 Fourier shell correlation threshold), which allowed first insights into its molecular architecture. Since sample preparations of 26S proteasomes are quite heterogeneous a high-throughput single particle data acquisition approach was pivotal to generate large data sets for an exhaustive classification [4,5].…”