The RssB response regulator directly targets sigma(S) for degradation by ClpXP - PubMed (original) (raw)
The RssB response regulator directly targets sigma(S) for degradation by ClpXP
Y Zhou et al. Genes Dev. 2001.
Abstract
The sigma(S) subunit of Escherichia coli RNA polymerase regulates the expression of stationary phase and stress response genes. Control over sigma(S) activity is exercised in part by regulated degradation of sigma(S). In vivo, degradation requires the ClpXP protease together with RssB, a protein homologous to response regulator proteins. Using purified components, we reconstructed the degradation of sigma(S) in vitro and demonstrate a direct role for RssB in delivering sigma(S) to ClpXP. RssB greatly stimulates sigma(S) degradation by ClpXP. Acetyl phosphate, which phosphorylates RssB, is required. RssB participates in multiple rounds of sigma(S) degradation, demonstrating its catalytic role. RssB promotes sigma(S) degradation specifically; it does not affect degradation of other ClpXP substrates or other proteins not normally degraded by ClpXP. sigma(S) and RssB form a stable complex in the presence of acetyl phosphate, and together they form a ternary complex with ClpX that is stabilized by ATP[gamma-S]. Alone, neither sigma(S) nor RssB binds ClpX with high affinity. When ClpP is present, a larger sigma(S)--RssB--ClpXP complex forms. The complex degrades sigma(S) and releases RssB from ClpXP in an ATP-dependent reaction. Our results illuminate an important mechanism for regulated protein turnover in which a unique targeting protein, whose own activity is regulated through specific signaling pathways, catalyzes the delivery of a specific substrate to a specific protease.
Figures
Figure 1
σS degradation by ClpXP and RssB in vitro. (A) Stimulation of ClpXP-dependent σS degradation by extracts of cells overproducing RssB. Degradation of [3H]σS by ClpXP was measured as described in Materials and Methods in the absence of a source of RssB (column 1); in the presence of 20 μg of an extract of E. coli MC4100 clpP− (Zhou and Gottesman 1998) carrying the plasmid vector pACYC184 (column 2); or in the presence of 20 μg of an extract of E. coli MC4100 clpP− carrying pUM-E, a pACYC184 derivative with a 5.7-kb _EcoR_I fragment that includes rssB (Rosl and Kersten 1994) (column 3). (B) Requirements for σS degradation with pure proteins. Degradation of [3H]σS was measured as described in Materials and Methods with purified components using 120 pmoles of [3H]σS. ClpP, ClpX, ATP, RssB, and acetyl phosphate were omitted where indicated.
Figure 2
RssB functions catalytically and specifically. (A) RssB acts catalytically. [3H]σS degradation was measured as a function of time as described in Materials and Methods in reaction mixtures with 2 pmoles of RssB, 120 pmoles of [3H]σS, 2 pmoles of ClpX, and 2 pmoles of ClpP. (B) RssB increases the apparent affinity for σS and increases the rate of the reaction. [3H]σS degradation was measured in reaction mixtures as described in Materials and Methods with 0.1 μM ClpX, 0.1 μM ClpP, and the indicated concentrations of σS, either with 0.4 μM RssB (filled circles) or without RssB (open circles). Reaction times were 2, 4, 6, and 10 min when RssB was included; and 10, 15, and 25 min in the absence of RssB. (C, D) RssB stimulates degradation of σS but not λ O. Degradation of [3H]σS (C) and [3H]λ O (D) was measured in reaction mixtures as described in Materials and Methods with varying amounts of ClpX and 19 pmoles of [3H]λ O substituted for [3H]σS in panel D, both with RssB (filled symbols) and without RssB (open symbols).
Figure 3
Interaction of σS and RssB. (A) RssB (150 pmoles) and [3H]σS (320 pmoles) were incubated in the absence of acetyl phosphate and analyzed by gel filtration in the absence of acetyl phosphate as described in Materials and Methods. (B) [3H]σS and RssB were incubated together with acetyl phosphate and analyzed by gel filtration in the presence of acetyl phosphate. (C) [3H]σS and RssB were incubated with acetyl phosphate for 10 min, treated with 20 mM EDTA for 1 h at 0°C, and then analyzed by gel filtration in the absence of acetyl phosphate and Mg2+. The positions where σS and RssB eluted when chromatographed separately, with or without acetyl phosphate, are indicated with arrows. Filled circles indicate the elution of σS, and open circles indicate RssB in A, B, and C. Molecular weight standards, Blue dextran 2000 (2000 kD), bovine serum albumin (67 kD), and chymotrypsinogen A (25 kD), eluted as indicated by arrows labeled void, 67, and 25, respectively.
Figure 4
Binding of σS to ClpX promoted by RssB. Reaction mixtures containing 9 pmoles of [3H]σS, 60 pmoles of ClpX, and the indicated amounts of RssB were incubated with ATP[γ-S] and acetyl phosphate as described in Materials and Methods. The mixtures were then centrifuged through microcon 100 ultrafiltration devices, and radioactivity in the retentates was measured as described. Complete (filled squares); minus ClpX (open circles).
Figure 5
Formation of ternary σS–RssB–ClpX complexes. (A) [3H]σS (215 pmoles), 150 pmoles of RssB, and 250 pmoles of ClpX were incubated in the presence of acetyl phosphate and ATP[γ-S] and analyzed by Sephacyl S-200 gel filtration as described in Materials and Methods. (B) σS was omitted. (C) RssB was omitted. The positions where ClpX, [3H]σS, and RssB eluted when chromatographed separately are indicated with arrows. σS is indicated by filled circles, RssB by open circles, and ClpX by dotted line. The positions where Blue dextran 2000 (2000 kD), bovine serum albumin (67 kD), ovalbumin (43 kD), and ribonuclease A (13.7 kD) eluted are indicated by arrows labeled void, 67, 43, and 13.7, respectively.
Figure 6
Formation of quaternary σS–RssB–ClpXP complexes and release of RssB with σS degradation. (A) [3H]σS (215 pmoles), 150 pmoles of RssB, 250 pmoles of ClpX, and 250 pmoles of ClpP were incubated in reaction mixtures containing acetyl phosphate and AMP-PNP in Buffer B and then analyzed by Sephacyl S-200 gel filtration as described in Materials and Methods. (B) Reactions were as in A, but ClpP was omitted. (C) [3H]σS, RssB, ClpX, and ClpP were incubated as in A, and complexes were isolated. Fractions containing σS–RssB–ClpXP complexes were pooled (fractions 29–31) and incubated in 200 μL with 10 mM ATP for 10 min at 24°C. Following incubation, the sample was chromatographed on the gel filtration column. In panel C, TCA-soluble radioactivity was measured in a portion of each fraction, and the radioactivity eluting between fractions 50 and 68 was soluble. The positions where ClpX, ClpP, σS, and RssB eluted when chromatographed separately are indicated with arrows. The positions where Blue dextran 2000 (2000 kD), bovine serum albumin (67 kD), ovalbumin (43 kD), and ribonuclease A (13.7 kD) eluted are indicated by arrows labeled void, 67, 43, and 13.7, respectively. In all three panels, the proteins were measured as described in Materials and Methods. σS is indicated by filled circles, RssB by open circles, ClpX by dotted line, and ClpXP by dashed line.
Figure 7
Competition between RssB and core RNA polymerase for σS binding. (A) Protection of σS from degradation by core RNA polymerase. [3H]σS (18 pmoles) alone (columns 1 and 2) or with 18 pmoles of σ70 (column 3) or 36 pmoles (column 4) of σ70 was added to 20-μL reaction mixtures containing Buffer B. Then, 18 pmoles of core RNA polymerase was added to reaction mixtures shown in columns 2, 3, and 4. After 20 min at 30 °C, RssB, ClpX, ClpP, acetyl phosphate, and ATP were added to all samples in a final volume of 25 μL as described for degradation assays in Materials and Methods. Following incubation for 30 min at 24°C, TCA-soluble radioactivity was measured. (B) Core binding by σS excludes RssB binding. [3H]σS (160 pmoles) and RssB (160 pmoles) were incubated for 20 min with acetyl phosphate as described in Materials and Methods for the formation of σS–RssB complexes. Then 160 pmoles of core RNA polymerase was added and after 10 min, the mixture was analyzed by Sephacryl S-100 gel filtration as described for the isolation of σS–RssB complexes. The positions where core, σS, and RssB eluted when chromatographed separately are indicated by arrows. Molecular weight standards, Blue dextran 2000 (2000 kD), bovine serum albumin (67 kD), and chymotrypsinogen A (25 kD), eluted as indicated by arrows labeled void, 67, and 25, respectively. The elution of σS (filled circles), RssB (open circles), and core (dashed line) are indicated.
Figure 7
Competition between RssB and core RNA polymerase for σS binding. (A) Protection of σS from degradation by core RNA polymerase. [3H]σS (18 pmoles) alone (columns 1 and 2) or with 18 pmoles of σ70 (column 3) or 36 pmoles (column 4) of σ70 was added to 20-μL reaction mixtures containing Buffer B. Then, 18 pmoles of core RNA polymerase was added to reaction mixtures shown in columns 2, 3, and 4. After 20 min at 30 °C, RssB, ClpX, ClpP, acetyl phosphate, and ATP were added to all samples in a final volume of 25 μL as described for degradation assays in Materials and Methods. Following incubation for 30 min at 24°C, TCA-soluble radioactivity was measured. (B) Core binding by σS excludes RssB binding. [3H]σS (160 pmoles) and RssB (160 pmoles) were incubated for 20 min with acetyl phosphate as described in Materials and Methods for the formation of σS–RssB complexes. Then 160 pmoles of core RNA polymerase was added and after 10 min, the mixture was analyzed by Sephacryl S-100 gel filtration as described for the isolation of σS–RssB complexes. The positions where core, σS, and RssB eluted when chromatographed separately are indicated by arrows. Molecular weight standards, Blue dextran 2000 (2000 kD), bovine serum albumin (67 kD), and chymotrypsinogen A (25 kD), eluted as indicated by arrows labeled void, 67, and 25, respectively. The elution of σS (filled circles), RssB (open circles), and core (dashed line) are indicated.
Figure 8
Model for the mechanism of action of RssB in regulating σS degradation by ClpXP. See text for discussion.
References
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