Volume 11, Issue 1, Pages 69-78 (January 2003) ATP Hydrolysis by the Proteasome Regulatory Complex PAN Serves Multiple Functions in Protein Degradation  Nadia Benaroudj, Peter Zwickl, Erika Seemüller, Wolfgang Baumeister, Alfred L Goldberg  Molecular Cell  Volume 11, Issue 1, Pages 69-78 (January 2003) DOI: 10.1016/S1097-2765(02)00775-X

Figure 1 Loosely Folded Substrates, Globular Substrates, and Peptides Activate ATP Hydrolysis by PAN to the Same Extent and Irrespective of the Presence of 20S Proteasomes (A) ATPase activity of PAN in the presence of loosely folded and globular substrates. 2.8 nM of PAN complex were incubated with 4 μM of casein, GFP, or GFPssrA as indicated at 45°C, and ATP hydrolysis was measured in 50 mM Tris (pH 7.5), 1 mM DTT, 1 mM ATP, and 10 mM MgCl2. Values are means plus standard deviations of three independent experiments. (B) Effect of 20S proteasomes on the basal and substrate-activated ATPase activity of PAN. 2.8 nM of PAN complex were incubated as in (A) in the presence of 14 nM of 20S proteasomes. (C) SsrA peptide inhibits unfolding of GFPssrA by PAN. The time course of fluorescence change of 500 nM of GFPssrA alone (•) or in the presence of 250 nM of PAN complex (▴) or 250 nM of PAN complex and 100 μM of ssrA peptide (○) was followed at 45°C in 50 mM Tris (pH 7.5), 1 mM DTT, 2 mM ATP, and 10 mM MgCl2. The initial decrease in GFPssrA fluorescence reflects the effects of 45°C on GFP fluorescence and does not indicate unfolding, which occurred only after addition of PAN and ATP. (D) SsrA peptide inhibits casein degradation by the PAN-20S complex. Degradation of 500 nM 14C-casein was followed with 2.1 nM of 20S proteasomes (open bars) or 2.1 nm 20S proteasomes and 9.3 nM of PAN complex (filled bars) in the presence of 550 μM of ssrA peptide as indicated at 45°C in 50 mM Tris (pH 7.5), 1 mM DTT, 1 mM ATP, and 10 mM MgCl2. Values are means plus standard deviations of three independent experiments. (E) SsrA peptide activates PAN's ATPase activity. ATP hydrolysis by PAN was measured without or with 4 μM of ssrA peptides as described in (A). Values are means plus standard deviations of three independent experiments. Molecular Cell 2003 11, 69-78DOI: (10.1016/S1097-2765(02)00775-X)

Figure 2 PAN Stimulates the Degradation of Globular and Unfolded Proteins by 20S Proteasomes The degradation of 1 μM of 14C-GFPssrA, acid-denatured 14C-GFPssrA, or 14C-casein was followed with 2.1 nM of 20S proteasomes alone (open bars), with 9.3 nM of PAN complex added (striped bars), or with 9.3 nM of PAN complex and 1 mM ATP added (filled bars) at 45°C in 50 mM Tris (pH 7.5), 1 mM DTT, and 10 mM MgCl2. Values are means plus standard deviations of three independent experiments. Molecular Cell 2003 11, 69-78DOI: (10.1016/S1097-2765(02)00775-X)

Figure 3 ATP Consumption by PAN during Degradation of Globular and Unfolded Proteins by 20S Proteasomes 1 μM of 14C-casein (upper panel), 14C-GFPssrA (middle panel), or acid-denatured 14C-GFPssrA (lower panel) were incubated with different concentrations of PAN (0, 1.54, 3.08, 4.62, 6.16, and 7.7 nM) and of 20S proteasomes (0, 7.14, 14,28, 21.42, 28.56, and 35.7 nM) at 45°C in 50 mM Tris (pH 7.5), 1 mM DTT, 1 mM ATP, and 10 mM MgCl2 in a 100 μl vol reaction. 50 μl were used to measure ATP hydrolysis, and 50 μl were used to measure protein degradation as described in Experimental Procedures. The rates of ATP hydrolysis were plotted against those of protein degradation. The data were fitted to a linear function, and the slope (shown in the lower right corner of each panel) was used to determined the number of moles of ATP hydrolyzed during degradation of one mole of protein. Values are means plus or minus standard deviations of three independent experiments. Molecular Cell 2003 11, 69-78DOI: (10.1016/S1097-2765(02)00775-X)

Figure 4 Deletion of Amino-Terminal Residues of 20S α Subunits Opens the Axial Channel of α Rings Electron microscopy top views of the α subunits of wild-type (A) and Δα(2-12) 20S proteasomes (B) were performed as described in Experimental Procedures. (C) represents the difference mapping of the averaged pictures. The diameter of both complexes is approximately 11 nm. Molecular Cell 2003 11, 69-78DOI: (10.1016/S1097-2765(02)00775-X)

Figure 5 Deletion of Amino-Terminal Residues of 20S α Subunits Increases Rate of Degradation of Unfolded Proteins and Eliminates Stimulation by PAN (A) Wild-type and Δα(2-12) 20S proteasomes degrade a tetrapeptide at similar rates. The degradation of 100 μM of Suc-LLVY-amc was carried out as described in Experimental Procedures with 420 nM of wild-type (solid line) or Δα(2-12) 20S (dashed line) at 45°C in 50 mM Tris (pH 7.5), 1 mM DTT, and 10 mM MgCl2. (B) Degradation of GFPssrA, acid-denatured GFPssrA, and casein by Δα(2-12) 20S. Degradation of 1 μM of 14C-radiolabeled protein was followed as in Figure 2. Values are means plus standard deviations of three independent experiments. (C) PAN promotes degradation of GFPssrA by the Δα(2-12) 20S. The time course of fluorescence change of 250 nM of GFPssrA was followed at 45°C in 50 mM Tris (pH 7.5), 1 mM DTT, and 10 mM MgCl2 in the presence of 20 nM of Δα(2-12)20S and 2 mM of ATP (•); or of 20 nM of Δα(2-12)20S, 10 nM of PAN complex, and 2 mM of AMPPNP (○); or of 20 nM of Δα(2-12)20S, 10 nM of PAN complex, and 2 mM of ATP (▴). Molecular Cell 2003 11, 69-78DOI: (10.1016/S1097-2765(02)00775-X)

Figure 6 After Unfolding of GFPssrA by PAN, Hydrolysis of ATP by PAN Is Still Essential for Its Degradation by the Δα(2-12)20S Proteasome Variant (A) The time course of fluorescence change of 250 nM of GFPssrA was followed in 50 mM Tris (pH 7.5), 1 mM DTT, 1 mM ATP, and 10 mM MgCl2 at 45°C in the presence of 50 nM of PAN (▵, •, ▴). After 15 min of incubation to allow unfolding of GFPssrA, 0 nM (▵) or 20 nM of Δα(2-12)20S was added to the samples together with 2 mM of ATPγS (•) or the equivalent volume of buffer (▵, ▴). If Δα(2-12)20S were present together with PAN and ATP from the onset (○), only buffer was added. (B) GFPssrA was incubated as in (A), and at the indicated times, aliquots were withdrawn, loaded on a 12% SDS-PAGE, and analyzed by Western blot using an anti-histidine antibody. The antibody also recognizes the β subunit of 20S proteasomes which carries a (His)6 tag. (C) GFPssrA amount on (B) was quantified and relative amounts were determined using the 0 time point as 100%. Relative amounts of GFPssrA in lanes 1–3 (○), 4–6 (▵), 7–9 (▴), and 10–12 (•) are plotted as indicated. Molecular Cell 2003 11, 69-78DOI: (10.1016/S1097-2765(02)00775-X)

Figure 7 Model for the Energy-Dependent Steps of PAN and Proteasomes-Mediated Protein Degradation Cut side views of 20S particles are schematized with the active sites represented as blue dots in the internal chamber. PAN is schematized as a hexameric ring. The circle and pentagonal shape of the subunits represent the substrate-free and substrate-bound forms of PAN, respectively. The gate which precludes entry of protein into 20S particles is represented in red. Molecular Cell 2003 11, 69-78DOI: (10.1016/S1097-2765(02)00775-X)