The RssB response regulator directly targets sigma(S) for degradation by ClpXP.
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.
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Y
Y
Zhou
S
S
Gottesman
J R
JR
Hoskins
M R
MR
Maurizi
S
S
Wickner
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D012808:Sigma Factor / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D012808:Sigma Factor; D014157:Transcription Factors; C098923:rssB protein, E coli; C069567:sigma factor KatF protein, Bacteria; D012321:DNA-Directed RNA Polymerases; D012697:Serine Endopeptidases; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases