Disassembly of the self-assembled, double-ring structure of proteasome α7 homo-tetradecamer by α6

Abstract

The 20S core particle of the eukaryotic proteasome is composed of two α- and two β-rings, each of which is a hetero-heptamer composed of seven homologous but distinct subunits. Although formation of the eukaryotic proteasome is a highly ordered process assisted by assembly chaperones, α7, an α-ring component, has the unique property of self-assembling into a homo-tetradecamer. We used biophysical methods to characterize the oligomeric states of this proteasome subunit and its interaction with α6, which makes direct contacts with α7 in the proteasome α-ring. We determined a crystal structure of the α7 tetradecamer, which has a double-ring structure. Sedimentation velocity analytical ultracentrifugation and mass spectrometric analysis under non-denaturing conditions revealed that α7 exclusively exists as homo-tetradecamer in solution and that its double-ring structure is disassembled upon the addition of α6, resulting in a 1:7 hetero-octameric α6-α7 complex. Our findings suggest that proteasome formation involves the disassembly of non-native oligomers, which are assembly intermediates.

Figure 1. Crystal structure of the human α7 homo-tetradecamer.

Ribbon models of the single and double α7 rings derived from the human α7 tetradecamer are shown in (a,b), respectively. Ribbon models of the α1–7 ring and the α1–7-β1–7 ring (half-proteasome) derived from the human 20S proteasome (PDB code 4R3O) are shown in (c,d), respectively. The left and right structures are related by a rotation of 90° around a horizontal axis. The α subunits are colored as follows: α1 (blue), α2 (green), α3 (magenta), α4 (forest green), α5 (orange), α6 (cyan), and α7 (red).

Figure 2. Characterization of the oligomeric state of the α7 subunit.

(a) Distribution of α7 sedimentation coefficients derived from SV-AUC experiments. The 14.2 S peak corresponds to the α7 homo-tetradecamer. (b) Mass spectrum of α7 under non-denaturing conditions. Red circles show the ion series of the α7 homo-tetradecamer.

Figure 3. Characterization of the oligomeric state of the α6 subunit.

(a) Distribution of α6 sedimentation coefficients derived from SV-AUC experiments. Inset shows the enlarged view with s-ranging from 0–10 displaying the peaks corresponding to monomer (2.6 S) and dimer (4.0 S) of α6. (b) Mass spectra of α6 at 5, 10, and 20 μM under non-denaturing conditions. Blue and orange circles show the ion series of the α6 monomer and dimer, respectively.

Figure 4. Characterization of the complex of α7 and α6 subunits.

(a) Distribution of sedimentation coefficients of mixtures of α7 and α6 at 1:0 (black line) and 1:4 (red line) molar ratios (α7 tetradecamer to α6 monomer) derived from SV-AUC experiments. The 10.2 S and 14.2 S peaks correspond to the 1:7 hetero-octameric complexes of α6 and α7 and the α7 homo-tetradecamer, respectively. (b) Mass spectra of mixtures of α7 and α6 at 1:0, 1:0.25, 1:0.5, 1:1, 1:2, and 1:4 molar ratios (α7 tetradecamer to α6 monomer). Magenta circles show the ion series of the 1:7 hetero-octamer complexes of α6 and α7.