Volume 22, Issue 2, Pages 193-204 (April 2006) Proteomic Profiling of ClpXP Substrates after DNA Damage Reveals Extensive Instability within SOS Regulon  Saskia B. Neher, Judit Villén, Elizabeth C. Oakes, Corey E. Bakalarski, Robert T. Sauer, Steven P. Gygi, Tania A. Baker  Molecular Cell  Volume 22, Issue 2, Pages 193-204 (April 2006) DOI: 10.1016/j.molcel.2006.03.007 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Quantitative MS Monitors ClpXP Substrate Recognition during DNA Damage (A) Cells containing ClpPtrap from untreated cultures grown in heavy (13C) leucine media and NAL-treated cultures grown in light media were mixed in equal numbers. After purification, ClpP-trapped proteins were digested with trypsin and analyzed by MS to identify and quantify peptides. (B) Incorporation control showing a peptide mass spectrum from E. coli grown in heavy leucine. The peptide sequence and m/z values for the light and heavy doubly charged ions (M + 2H)2+ are displayed above the spectrum. No peaks corresponding to a light leucine peptide (one or two light leucines) are seen above background (see inserts, magnified 1000×), demonstrating complete incorporation of 13C leucine. (C) Distribution of SILAC ratios (light/heavy) for the constant-time trapping experiment. Proteins with SILAC ratios varying more than 3-fold in the constant-time and constant-density trapping experiments have thick outlines. The symbol shape denotes transcriptional change after DNA damage based on published microarray experiments; gray circles, ≥1.5-fold increase (LexA-dependent [SOS] and independent); black squares, ≥ 1.5-fold decrease. Molecular Cell 2006 22, 193-204DOI: (10.1016/j.molcel.2006.03.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 ClpXP Degrades RecN and UvrA (A) RecN degradation in vivo, assayed by Western blotting after inhibition of protein synthesis. Substrates had N-terminal epitopes. Symbols are black for clpX+, gray for clpX−, squares for 50 μM NAL, and triangles for no NAL. RecN with a C-terminal AA → DD mutation was stable (DD in insert). (B) Degradation of UvrA in vivo. Methods and symbols as in (A). (C) ClpXP degradation in vitro of 0.5 μM GFP-RecN or RecN assayed by SDS-PAGE. (D) Michaelis-Menten plot of ClpXP degradation of GFP bearing the last seven residues of RecN (GFP-RecN; open symbols) or wild-type RecN (closed symbols). The rates at each protein concentration were determined three times, and the average and standard deviations are plotted. For GFP-RecN, the curve is a fit with KM = 1.2 μM; Vmax = 1.1 molecules/minute/ClpX6. Molecular Cell 2006 22, 193-204DOI: (10.1016/j.molcel.2006.03.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Degradation of Additional Substrates In Vivo (A) ClpX-dependent degradation of epitope-tagged YebG and DinD, and DinI. (B) Western blot of SulA protein associated with ClpPtrap purified from clpX−clpA− cells (left), clpX+ cells (middle), or clpX+ cells treated with 50 μM NAL (right). (C) Left, ClpX-dependent degradation of MinD. Degradation rates did not change upon treatment with 50 μM NAL. Right, ratio of intracellular MinD levels ± NAL determined by Western blot (error ± SD [n = 3]). The SILAC ratio is shown for comparison. In (A) and (C), degradation was assayed by Western blot after inhibiting protein synthesis. Molecular Cell 2006 22, 193-204DOI: (10.1016/j.molcel.2006.03.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Uncorrelated Changes in Cellular Levels and Trapping (A) Ratios of cellular Dps and σS levels (±50 μM NAL) were determined by Western blotting (σS) or immunoprecipitation (Dps) in clpP+ and clpP− cells. (B) Degradation of σS was measured by Western blotting after inhibition of protein synthesis; black squares, 50 μM NAL; gray circles, no NAL. (C) Western blots show that cellular levels of RssB and SspB are not affected by addition of 50 μM NAL. In all cases, the values are an average of at least three determinations ± SD. Molecular Cell 2006 22, 193-204DOI: (10.1016/j.molcel.2006.03.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Effects of DNA Damage versus Substrate Overexpression on Trapping (A) Flow chart of experimental design. (B) Dps and σS levels in ClpPtrap decrease after treatment with NAL, as determined by Western blot. Overproduction of RecN did not decrease the amount of Dps or σS trapped. RecN-AA with the wild-type ClpX-recognition signal was trapped efficiently, whereas RecN-DD was trapped very poorly. (C) Autoradiograph of 2D gels of substrates captured by ClpPtrap after pulse labeling cultures, ±NAL treatment. As expected from the MS data, some spots were more abundant after NAL treatment (examples labeled with arrows). Spots representing Dps and σS are also marked; their positions were inferred based on pI, molecular weight, and previous 2D gels. Molecular Cell 2006 22, 193-204DOI: (10.1016/j.molcel.2006.03.007) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Highly Induced SOS Proteins Are More Likely to Be Rapidly Degraded (A) Based on published microarray data, estimates of the number of SOS regulon members range from 30 to 50 and, of these, eight are highly (>8-fold) induced. Most of these proteins have known roles in the SOS response and are rapidly degraded. (B) Some of the ten proteins with medium induction ratios (3- to 8-fold) are also rapidly degraded. Little is known about the stability of the SOS proteins with modest (<3-fold) induction. Molecular Cell 2006 22, 193-204DOI: (10.1016/j.molcel.2006.03.007) Copyright © 2006 Elsevier Inc. Terms and Conditions