Volume 7, Issue 3, Pages 627-637 (March 2001) ATP-Dependent Proteases Degrade Their Substrates by Processively Unraveling Them from the Degradation Signal  Cheolju Lee, Michael P Schwartz, Sumit Prakash, Masahiro Iwakura, Andreas Matouschek  Molecular Cell  Volume 7, Issue 3, Pages 627-637 (March 2001) DOI: 10.1016/S1097-2765(01)00209-X

Figure 1 Schematic Drawings of Protease Substrate Proteins Three-dimensional structures of barnase ([A]), E. coli DHFR ([B]), and a linear representation of chimeric proteins ([C]) are shown. Tight-binding ligands barstar and methotrexate are shown in dark gray. In the circular permutants of E. coli DHFR, the original N and C termini are linked by a five glycine linker, and new N termini are created at Pro-25, Lys-38, Asp-70, and Ala-145. The positions of newly created N and C termini are shown in black. Figures were produced by RasMol (Sayle and Milner-White, 1995) Molecular Cell 2001 7, 627-637DOI: (10.1016/S1097-2765(01)00209-X)

Figure 2 Degradation of N Terminally–Tagged DHFR and Barnase by ClpAP and Proteinase K Degradation reactions were carried out at 25°C with no ligand (circles) and with 100 μM barstar for barnase substrates or 10 μM methotrexate for DHFR substrates (squares). The vertical axis represents the amount of undegraded substrate protein as a percentage of the total amount of substrate added at the beginning of the experiment. Curves through the data points are the best fits to a single exponential or a straight line Molecular Cell 2001 7, 627-637DOI: (10.1016/S1097-2765(01)00209-X)

Figure 3 Degradation of N Terminally–Tagged Circular Permutants of E. coli DHFR by ClpAP and Proteinase K (A) Conditions were as in Figure 2. Degradation was performed in the absence (circles) or presence (squares) of 10 μM methotrexate. (B) To determine the dependence of degradation by ClpAP on temperature, experiments were performed at 25°C (circles) and 10°C (squares) Molecular Cell 2001 7, 627-637DOI: (10.1016/S1097-2765(01)00209-X)

Figure 4 Degredation of Proteins by the Proteasome and ClpXP (A) Mouse DHFR and barnase were degraded by the proteasome at 25°C in the absence of ligand (circles) or in the presence (squares) of 1 μM methotrexate for DHFR substrates or 10 μM barstar for barnase substrates. Degradation of E. coli DHFR and its circular permutants was performed in the absence of ligand (circles) or in the presence (squares) of 100 μM methotrexate. (B) Degradation of C terminally–tagged substrate proteins by ClpXP at 30°C in the absence of ligand (circles) or in the presence (squares) of 1 μM barstar for barnase substrates or 10 μM methotrexate for DHFR substrates Molecular Cell 2001 7, 627-637DOI: (10.1016/S1097-2765(01)00209-X)

Figure 5 Sequential Degradation of Two-Domain Proteins (A–D) Proteasome degradation of a DHFR-barnase fusion protein ([A] and [B]) or a barnase-DHFR fusion protein ([C] and [D]) in the presence of 1 μM barstar ([A] and [C]) or 10 μM methotrexate ([B] and [D]). The left panels show autoradiograms of SDS–PAGE gels. The T lane contains the untreated ubiquitin-fusion protein (indicated by arrowheads). In the zero time lane, the N-terminal ubiquitin moiety has been completely removed during a preincubation in ATP-depleted reticulocyte lysate, leaving the two domain proteins with N-terminal linker (indicated by arrows). Quantification of the autoradiograms is shown in the graphs in the right panels. The amount of remaining substrate proteins including their ubiquitinated forms (circles) or the amount of the degradation end product (squares) is plotted. (E) Estimate of the molecular weight of the degradation end product (closed square) in (C) and (D) by SDS–PAGE. Molecular weight of the degradation end product was estimated by comparing the relative mobility of the end product (closed square) to those of standard proteins (open squares). Relative mobilities were plotted against logarithms of the number of amino acid residues. The number of amino acid residues of the molecular weight standards are 150, 202, 226, 233, 278, 309, 317, and 393. (F) Sequential degradation of two-domain substrate proteins by ClpAP and ClpXP. The experimental design was as for (A)–(D), but only one time point is shown. Barnase and DHFR were fused in-frame, and an N-terminal degradation tag was attached for ClpAP experiments, or the C-terminal SsrA tag was attached for ClpXP experiments. Specifically, the constructs are: lane 1, N-terminal tag/barnase/DHFR; lane 2, N-terminal tag/DHFR/barnase; lane 3, barnase/DHFR/SsrA tag; and lane 4, DHFR/barnase/SsrA tag. Substrate proteins were incubated with ClpAP (lanes 1 and 2) or ClpXP (lanes 3 and 4) in the presence of the specified ligand at 25°C for 30 min for ClpAP or 120 min for ClpXP and analyzed by SDS–PAGE and autoradiography. Partially processed products are marked by arrowheads, and their molecular sizes were estimated as in (E). Lane M shows molecular weight markers. MTX, methotrexate Molecular Cell 2001 7, 627-637DOI: (10.1016/S1097-2765(01)00209-X)

Figure 6 Processing of p105 (A) Organization of domains in the NF-κB precursor p105. REL, Rel homology domain; NLS, nuclear localization signal; GRR, glycine rich region; Ubi, ubiquitination domain; AR × 7, seven ankyrin repeats; and PEST, PEST sequence. The locations of the analyzed mutations are shown, as is the processing point at which degradation by the proteasome stops. (B) Processing of p105 to p50 by the proteasome. Wild-type p105 and the mutants Glu-290→Ala, Phe-348→Ala, and Tyr-350→Ala were incubated in reticulocyte lysate at 37°C. At the indicated times, samples were taken and analyzed by SDS–PAGE and autoradiography. The positions of bands representing p105 and p50 are indicated by arrows. p50 accumulates in the processing of wild-type and mutant Glu-290→Ala, but not for the mutants Phe-348→Ala and Tyr-350→Ala. (C) Degradation of wild-type and mutant p105. The graph plots the amount of full-length p105 for wild type (circles) as well as the mutants Phe-348→Ala (squares) and Tyr-350→Ala (diamonds) as a function of processing time. Data were obtained by quantifying the radioactivity in the bands representing p105 in the gels shown in (B). (D) Processing of wild-type p105 and the mutants Phe-31→Ala, Glu-290→Ala, Phe-348→Ala, Tyr-350→Ala, and Phe-371→Ala. Experimental procedures are as for (B), but only the 3 hr time point is shown Molecular Cell 2001 7, 627-637DOI: (10.1016/S1097-2765(01)00209-X)