The proteasome under the microscope: the regulatory particle in focus

Abstract

Since first imaged by electron microscopy, much effort has been placed into determining the structure and mechanism of the 26S proteasome. While the proteolytic core is understood in atomic detail, how substrates are engaged and transported to this core remains elusive. Substrate delivery is accomplished by a 19-subunit regulatory particle that binds to ubiquitinated substrates, detaches ubiquitin tags, unfolds the substrate, and translocates it into the peptidase in an ATP-dependent fashion. Recently, several labs have determined subnanometer cryoEM structures of the 26S proteasome, shedding light on the architecture of the regulatory complex. We discuss the biological insights into substrate processing provided by these structures, and the technical hurdles ahead to achieve an atomic resolution structure of the 26 proteasome.

Figure 1

Proteasome architecture. A. Locations of the base (blue), lid (green), and ubiquitin receptors Rpn10 and Rpn13 within the proteasomal RP. While Rpn13 is considered to be part of the base subcomplex, Rpn10 attaches primarily to the lid and stabilizes the lid-base interaction. B. Atomic models of the RP and the CP fit into the subnanometer reconstruction, shown on the left in the same orientation as A, and facing the lid component on the right (PDB ID 4b4t, except for Rpn1 and Rpt1–6, which were provided by the Pablo Chacón lab). C. The interactions between the Rpts (blue) and Rpn1 and Rpn2 (purple) subunits of the base subcomplex of the RP are shown. D. Architecture of the lid subcomplex. On the left, the horseshoe arrangement of the PCI domains is highlighted in black. On the right, the lid is viewed from the top down, showing the MPN heterodimer (red and green), and the bundle of C-terminal helices (outlined by a black oval). The reconstruction accession number used for this figure is EMD-1992.

Figure 2

Comparison of segmented subunits from three subnanometer reconstructions. Density maps for two RP subunits, Saccharomyces cerevisiae Rpn2 and Drosophila melanogaster Rpn6, were generated from their crystal structures (PDB IDs: 4ady and 3txn, respectively) and filtered to 7Å resolution. The first 37 residues of Rpn6, which are predicted to form two short alpha helices, are absent from the crystal structure and thus do not appear in the simulated 7Å density. A gray ellipse (outlined in red) is used to represent these N-terminal helices. The densities corresponding to the Rpn2 and Rpn6 subunits were segmented from the subnanometer-resolution reconstructions of proteasomes from Saccharomyces cerevisiae (EMD-1992 and EMD-2165) and Homo sapiens (EMD-2047). The segmented densities were aligned to the simulated EM density and a cross-correlation value calculated.

Figure 3

Rearrangements of the lid subunits upon incorporation into the RP. The atomic models of the lid subunits (PDB ID: 4b4t) were docked into reconstructions of the isolated lid (EMD-1993) and holoenzyme (EMD-1992). The N-terminal region of Rpn9 (purple) was duplicated to occupy the N-terminal region of Rpn3 (dark yellow), for which there is no atomic model. The structures are shown from the top (A), front (B), and side (C). Several subunits, in particular the N-terminal arm of Rpn5, undergo considerable movements between the isolated and integrated states. In the isolated form, the Rpn5 N-terminal helices are folded up against Rpn11, potentially blocking the DUB active site, which is located at the bottom of Rpn11 and facing Rpn5. Upon lid binding, the Rpn5 N-terminal arm swings down to interact with the CP, and the Rpn8/Rpn11 heterodimer (red and green) extends toward the center of the RP.

Figure 4

Model for substrate degradation and staircase arrangement of the ATPase. A. Putative model for ATP-dependent substrate deubiquitination and degradation. (i) Binding of the Rpn10 UIM (yellow cylinder) between two ubiquitin moieties of a tetraubiquitin chain (purple). (ii) The unfolded tail of the substrate (red) is threaded through the ATPase pore and becomes engaged. At this point, the isopeptide bond between the substrate and the tetraubiquitin chain is not in the vicinity of the DUB active site (pink, circled in yellow), which is located at the bottom of Rpn11 (green) and faces the ATPase pore. (iii) Translocation of the substrate tail progresses in an ATP-dependent fashion, leading to the positioning of the isopeptide linkage between the ATPase pore and the DUB active site, and the ubiquitin chain is cleaved off. (iv) As the tetraubiquitin dissociates, the remainder of the substrate is unfolded and translocated into the peptidase for degradation. B. The heterohexameric arrangement of the ATPase catalytic domains (light and dark blue) are shown atop the CP (grey), looking down the central pore (EMD-2165). C. The segmented densities corresponding to the catalytic domains are lined up with their exterior surface facing the reader. The large AAA+ subdomains become progressively more upright, as indicated by the red dashed line, producing a staircase-like arrangement in the closed ATPase ring. Interestingly, Rpt6 is suspended above the CP surface at an intermediate height between Rpt3 and Rpt2.