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.

Fig. 1.

Structure of the 26S proteasome from S. pombe. Model of the 26S proteasome and two different views rotated around the pseudo-sevenfold axis of the CP by 90°. The isosurface threshold was set to include a protein mass of 755 kDa for the CP. Secondary structure elements like bihelical repeats can be clearly discerned (A, *). B shows the isosurface representations of the model (lower half). The transparent view through the reconstruction shows the fitted atomic models of the CP (red, **: α-helix of subunit α4) and the AAA-ATPase (blue). A mesh representation of the reconstruction with an overlay isosurface in green highlighting the main variances among the particles is shown in C.

Fig. 2.

Classification of S. pombe 26S proteasomes reveals variations in the RP. Five class averages obtained by focused classification (CP in red, AAA-ATPase-ring in blue, remaining RP in brown) are shown in A. Major differences of each class to the overall average correlate to the localization of the variance map shown in Fig. 1 (B) Additional density in green; (C) missing density in yellow. As expected from ML3D classification, a subset of 26S proteasomes is lacking the extra mass (class 4). Further classes show additional densities in the region between base and lid, close to the upper AAA-ATPase ring (classes 2, 3, and 5).

Fig. 3.

Monoclonal anti-FLAG antibodies bind to the Rpn11–C-terminal 3xFLAG-tag in intact 26S proteasomes. After incubation of proteasomes with anti-FLAG-antibody, extra densities are clearly visible in 2D class averages (A, Top Left: control). After classification and 3D reconstruction (B) the center of mass of the antibody can be clearly seen on the top of the RP lid (green). The cross-link between Lys49 in the coiled-coil region of Rpt3 (C, yellow) and Lys281 of Rpn11 has an estimated length of 20 Å and allows mapping Rpn11:Lys281 within a sphere of 20-Å radius (yellow sphere). A sphere of ∼80-Å radius (dotted line) with its central point in the center of mass of the antibody depicts the possible localization of the IgG bindings site.

Fig. 4.

Structural and mechanistic depiction of the regulatory particle. Rpt1-α4 and Rpt6-α2 cross-links (A, yellow) between AAA-ATPase (blue) and CP (red) corroborate the previously suggested CP-AAA topology (32). The cross-links between AAA-ATPase subunits (A, green) confirm the Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5 topology of the AAA-ATPase hexamer. Cross-links in the AAA cavity are indicative of inducible conformational changes of central rings (A, magenta). (B) Top-view visualizing the positioning of the AAA-ATPase ring (t1–t6, center of AAA ring: white asterisk) relative to the α-ring of the CP (α1–α7, center: black asterisk). (C) Integrated model of the RP and α-ring of the CP: Ubiquitylated substrates are recognized at the RP (e.g., Rpn10 or Rpn13) of the 26S proteasome, deubiquitylated (e.g., Rpn11 or Uch2) for Ub recycling and translocated (Rpt1–6) through the AAA-ATPase cavity and the opened gate (Rpt2, Rpt5) of the CP for degradation (extra mass, cyan; Rpt1–6, as in B; Rpt3–Rpn11 cross-link, as in Fig. 3).