The RssB response regulator directly targets σS for degradation by ClpXP

Abstract

The σS subunit of Escherichia coli RNA polymerase regulates the expression of stationary phase and stress response genes. Control over σS activity is exercised in part by regulated degradation of σS. 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 σS in vitro and demonstrate a direct role for RssB in delivering σS to ClpXP. RssB greatly stimulates σS degradation by ClpXP. Acetyl phosphate, which phosphorylates RssB, is required. RssB participates in multiple rounds of σS degradation, demonstrating its catalytic role. RssB promotes σS degradation specifically; it does not affect degradation of other ClpXP substrates or other proteins not normally degraded by ClpXP. σS 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[γ-S]. Alone, neither σS nor RssB binds ClpX with high affinity. When ClpP is present, a larger σS-RssB-ClpXP complex forms. The complex degrades σS 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.