Molecular architecture and assembly of the eukaryotic proteasome

Abstract

The eukaryotic ubiquitin-proteasome system is responsible for most aspects of regulatory and quality-control protein degradation in cells. Its substrates, which are usually modified by polymers of ubiquitin, are ultimately degraded by the 26S proteasome. This 2.6-MDa protein complex is separated into a barrel-shaped proteolytic 20S core particle (CP) of 28 subunits capped on one or both ends by a 19S regulatory particle (RP) comprising at least 19 subunits. The RP coordinates substrate recognition, removal of substrate polyubiquitin chains, and substrate unfolding and translocation into the CP for degradation. Although many atomic structures of the CP have been determined, the RP has resisted high-resolution analysis. Recently, however, a combination of cryo-electron microscopy, biochemical analysis, and crystal structure determination of several RP subunits has yielded a near-atomic-resolution view of much of the complex. Major new insights into chaperone-assisted proteasome assembly have also recently emerged. Here we review these novel findings.

Figure 1. Schematics of the ubiquitin-proteasome system and a AAA+ chambered protease

A, Through the sequential actions of E1, E2, and E3 enzymes, a protein to be degraded is modified with a polyubiquitin chain, which serves as a targeting signal for the proteasome. B, A cutaway view of a AAA+ chambered protease, displaying the path of substrates through the ATPase ring and into the proteolytic chamber. The width of the passage into the protease chamber is delineated by yellow lines, and the proteolytic active sites are shown as red dots. In this protease, the catalytic chamber is bracketed by two antechambers. Adapted with permission from Nature Publishing Group, copyright 2012.

Figure 2. Structure of the 26S proteasome

A. The cryo-EM density of the 26S proteasome is shown. The 19S regulatory particle (RP) lid subcomplex is displayed in yellow, the RP base subcomplex in blue, and the 20S core particle (CP) in grey. Known and putative assembly chaperones for each subcomplex are displayed to the right. Adapted with permission from Nature Publishing Group (46), copyright 2011. B, Space-filling model of the 20S CP atomic structure from yeast (PDB ID = 1RYP). C, A view into the axial pore of the 20S CP from T. acidophilum. In this model (PDB ID = 3IPM), the archaeal ATPase HbYX motif (red ribbons) are inserted into the pockets formed at the interfaces of two adjacent α subunits, opening the CP gate. D, the HbYX motif interfaces with a critical lysine residue (shown in yellow) in the α pocket.

Figure 3. Organization of the RP, with atomic and pseudoatomic models of base subunits

A bird’s eye view of the 26S proteasome EM density is shown. Where available, the atomic or pseudoatomic models of Rpt1-6 (modeled using PDB ID = 3H4M, 3H43), Rpn2 (PDB ID = 4ADY), Rpn10 von Willebrand domain (PDB ID = 2×5N), and Rpn13 PRU domain (PDB ID = 2R2Y) are shown and colored the same as their respective EM densities. Adapted with permission from Proceedings of the National Academy of Science of the U.S.A (47), copyright 2012.

Figure 4. Proteasomal AAA+ ATPase structure and hexameric ring organization

A. The domain architecture of the M. jannaschii proteasome-activating nucleotidase (PAN) is shown. The eukaryotic ATPases Rpt1-6 share a similar domain organization as PAN. B. A pseudoatomic model of the proteasomal ATPase ring (modeled as in Figure 3) is shown. Domains are colored as in (A) C. Arrangement of the eukaryotic ATPases in the heterohexameric ring. The inferred proline cis/trans isomerism of each Rpt subunit is listed. Subunits forming pairs in the “trimer of dimers” model are similarly colored. See text for details.

Figure 5. 19S base ubiquitin receptor and scaffold subunit architecture

A. Ribbon structure of Rpn2 from S. cerevisiae (PDB ID = 4ADY). The N-terminal helical domain is colored blue; the toroidal domain green; and the C-terminal domain red. B. An axial view of the Rpn2 toroid, illustrating the central α helices. PC repeats are colored blue (N-terminal) through orange (C-terminal), with the central helices in red. C. Domain organization of the intrinsic ubiquitin receptors Rpn10 and Rpn13, and the extrinsic receptors Rad23, Dsk2, and Ddi1. Domains and amino acid numbering is according to the S. cerevisiae gene products. vWA, von Willebrand factor A domain; UIM, ubiquitin-interacting motif; PRU, Pleckstrin-like receptor for ubiquitin domain; Ubl, ubiquitin-like domain; UBA, ubiquitin-associated domain. D. Distinct modes of interaction with Ub are utilized by each ubiquitin receptor. Amino acids of each Ub-binding domain (shown in grey) that contact Ub (green) are highlighted in red. (PDB ID = 2Z59, 2KDE, 1WR1 for PRU, UIM, and UBA, respectively).

Figure 6. Molecular architecture of the proteasome lid

A. The EM structure of the purified lid complex and the densities contributed by each subunit are shown. B. Atomic structure of the D. melanogaster Rpn6 PCI domain PDB ID = 3TXN), illustrating the N-terminal helical region and the C-terminal winged helix domain. C. Atomic structure of the human Mov34 MPN domain (PDB ID = 2O95). Adapted with permission from Nature Publishing Group (46), copyright 2011.

Figure 7. The 20S CP assembly pathway

See text for details.

Figure 8. Assembly of the RP base is mediated by RP assembly chaperones

A, The RP base assembly pathway in yeast. See text for details. B, C. Interaction between (B) S. cerevisiae Nas6 and the C-domain of Rpt3 (PDB ID = 2DZN) and (C) S. cerevisiae Hsm3 and the Rpt1 C-domain (PDB ID = 3VLF). C-domains are similarly oriented. D. Atomic structure of S. cerevisiae Rpn14 (PDB = 3ACP).

Figure 9. Assembly and attachment of the RP lid

A, The RP lid assembly pathway inferred from studies in yeast. MPN subunits are shown in purple, PCI domain subunits are shown in peach, and Sem1 is shown in light blue. See text for details. B, A model for the stabilization of lid-base interaction by Rpn10 and the Rpn12 C-terminal tail via interaction with Rpn2 during RP assembly. In this model, Rpn10 positions Rpn11 to interact with Rpn2 (curved arrow), while the C-terminal tail of Rpn12 directly contacts the Rpn2 N-terminal extension (straight arrow). The N- and C-termini of Rpn2 are marked.