Complete subunit architecture of the proteasome regulatory particle

Abstract

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.

Figure 1. The lid subcomplex within the holoenzyme assembly

a) Negative-stain 3D reconstruction at ~15 Å resolution shows resemblance between endogenous (left) and recombinant (right) lid. b) Locations of lid (yellow) and base (cyan) within the subnanometer holoenzyme reconstruction. c) Six copies of the crystal structure of a PCI domain (PDBid: 1RZ4) are docked into the lid electron density, showing a horseshoe-shaped arrangement of the winged-helix domains. Each domain is colored according to its respective lid subunit (Fig. 2).

Figure 2. Three-dimensional reconstructions of the recombinant lid subcomplex and the yeast 26S proteasome

a) Negative stain reconstruction of the isolated lid subcomplex at 15Å resolution, colored by subunit and shown from the exterior (left), the side (middle), and the interior, base-facing side (right). A dotted line (middle) indicates the highly variable electron density for the flexible N-terminal domains of Rpn5 and 11. b) Subnanometer cryoEM reconstruction of the holoenzyme, shown in three views corresponding to the isolated lid and colored as above, with the core particle in grey.

Figure 3. Localization of Rpn1 and Rpn2, and ubiquitin-interacting subunits

a) Rpn1 (top) and Rpn2 (bottom) are oriented to emphasize similarities in their domain structure and solenoid attachment to the extended N-terminal helices of Rpt1 and Rpt3/6, respectively. b) Side and top views of the regulatory particle, showing the locations of the ubiquitin receptors Rpn10 and 13, and the DUB Rpn11 relative to the central pore. Crystal structures for Rpn10 (PDBid: 2×5n), Rpn13 (PDBid: 2r2y), and an MPN domain homologous to Rpn11 (AMSH-LP, PDBid: 2znr) are shown docked into the EM density. The predicted active site of Rpn11 is indicated (red dot).

Figure 4. Conformational rearrangements of the lid subcomplex upon integration into the holoenzyme

The lid complex in its isolated (left) and integrated (right) state is shown as viewed from the exterior (a) and top (b) of the regulatory particle. Major subunit rearrangements are depicted by arrows. The N-terminus of Rpn5 (light yellow) interacts with Rpn11 in the isolated complex, and swings down to contact the core particle upon incorporation into the holoenzyme. The N-terminal domain of Rpn6 swings to the left to interact similarly with the core particle. Rpn3, 8, and 11 undergo dramatic rearrangements, in which they move towards the center of the regulatory particle.

Figure 5. Structural features of the base ATPase subunits

a) Positions of Rpt2 (cyan), 3 (green), and 5 (orange) within the base hexameric ring and relative to the 20S core (grey) are shown using fitted crystal structures of the homologous PAN AAA+ domain (PDBid: 3h4m). The EM density contains the molecular envelope of the C-terminal tails (dark blue), docked into their cognate binding sites on the 20S core. Corresponding densities were not found for the tails of the Rpt1, 4, and 6 (grey ribbon structure). b) Cutaway side view of the holoenzyme EM density with Rpt1-5 visible. Individually docked copies of the PAN crystal structure reveal a spiral staircase arrangement of the Rpts, emphasized by space-filling representations of the PAN pore-1 loop residues (not resolved in the Rpts).

Figure 6. Model for the recognition, deubiquitination, and engagement of a poly-ubiquitinated substrate by the 26S proteasome

A K48-linked tetra-ubiquitin chain (magenta, PDBid: 2kde) is conjugated to the unstructured initiation region of a substrate (red) and bound to the ubiquitin receptor Rpn13 (orange). The substrate is poised for deubiquitination by Rpn11 (green, active site indicated by star), and its unstructured initiation region is engaged by the translocation machinery of the base (cyan). A poly-ubiquitin chain could alternatively bind to the UIM of Rpn10 (yellow) or interact with both receptors simultaneously. The DUB Ubp6 is localized further from the central pore, in a position to trim excess ubiquitin chains.