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 ςSdegradation 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.