For many years, our lab has investigated the role of energy-dependent proteolysis in regulation of gene expression in bacteria. The ATP-dependent cytoplasmic proteases, akin to the eukaryotic proteasome, contain ATPase domains or subunits that recognize substrates and unfold them, feeding them to the proteolytic domains. Bacteria contain multiple ATP-dependent proteases; five of them have been characterized in E. coli. Abnormal or misfolded proteins are degraded by these proteases. In addition to this quality control role, the proteases degrade proteins that are naturally unstable; for these proteins, degradation is likely to play an important biological role. Such protease substrates fall into two general classes: proteins that are always degraded, so that regulation of their abundance depends primarily on changes in synthesis, and proteins that show regulated proteolysis. In all cases, identifying how the substrate is recognized by the protease and how recognition is affected by growth conditions is important in understanding how regulation is carried out. In the past, our lab showed that the Lon ATP-dependent protease regulated capsular polysaccharide synthesis and cell division by degrading the RcsA and SulA proteins, discovered and characterized the two-component Clp proteases, ClpAP and ClpXP, and investigated the roles of these proteases in vivo and in vitro. In recent years, our focus has been on the regulated degradation of the RpoS sigma factor, a subunit of RNA polymerase that directs the polymerase to specific promoters. RpoS is important for cells to switch to a stationary or stress response gene expression program. The cell regulates RpoS accumulation in a variety of ways, including at the level of translation via small RNA activators of translation, and by regulated proteolysis. We have been studying this proteolysis, one of the best examples of regulated protein turnover in E. coli. RpoS is rapidly degraded during active growth, in a process that requires the energy-dependent ClpXP protease and the adaptor protein RssB, a phosphorylatable protein that presents RpoS to the protease. RpoS becomes stable after various stress or starvation treatments; the mode of stabilization was a mystery until work from our lab led to discovery of a small, previously uncharacterized protein, now named IraP (inhibitor of RssB activity after phosphate starvation). Mutants of iraP abolish the stabilization of RpoS after phosphate starvation. IraP blocks RpoS turnover in a purified in vitro system, and directly interacts with RssB. In E. coli, phosphate starvation is sensed by an increase in the levels of the small molecule ppGpp, and the iraP promoter is positively regulated by ppGpp. Two other small proteins also stabilize RpoS in a purified in vitro system, IraM, and IraD. These proteins are not similar in predicted structure to IraP. IraM is made in response to magnesium starvation, dependent on the PhoP and PhoQ regulators; IraD is important after DNA damage. The anti-adaptors define a new level of regulatory control, interacting with the RssB adaptor protein and blocking its ability to act; environmental signals regulate RpoS turnover by regulating expression of different anti-adaptors. We continue our studies on the structure and function of RssB and its anti-adaptors. We use genetic selections to identify mutations in RssB resistant to a specific anti-adaptor, define interactions between wild-type and mutant versions of the antiadaptors and RssB using a bacterial two-hybrid system and, in collaborative studies investigate the structures of these proteins and their in vitro function. This is a long-term collaboration with the lab of Sue Wickner (NCI). The N-terminal domain of RssB is a member of the widespread response regulator family. Collaborative studies with X. Ji (NCI) on the structure of IraP reveal that it is a unique protein with similarity to B-Zip dimers. Mutational and biochemical analysis of mutants inboth IraP and RssB are defining how these interact and providing insight into how RssB works to deliver RpoS to the protease. We are collaborating with A. Deaconescu (Brown University), who has solved the structure of an IraD/RssB complex, providing valuable new insight into how IraD inactivates RssB and fully supporting our earlier genetic and biochemical studies. She is currently extending her studies to IraM and IraP as well. IraM interacts with C-terminal domain of RssB; this domain has homology to an inactive PP2C phosphatase domain. One class of RssB mutations that are resistant to all of the anti-adaptors activates RssB, bypassing the stimulatory effect of phosphorylation. These mutants provide new insight into how RssB works and how regulatory proteins can disrupt the function of the conserved domains that make up RssB. We are further defining how RssB interacts with ClpX, the ATPase subunit of the ClpXP protease. The N-terminal domain of ClpX, known to interact with some other adaptors and substrates, interacts with the RssB C-terminus. Continued dissection of this system is providing insight into how this process is balanced in the cell. Other anti-adaptors are likely to exist, based on a variety of results. The bacterial two-hybrid system has been used to identify other proteins that interact with RssB. Interacting proteins may be additional anti-adaptors or other substrates for RssB. One partner, AnmK, has been studied in some detail. AnmK is an enzyme involved in recycling of peptidoglycan. Overexpression of AnmK stabilizes RpoS, most consistent with it acting as a novel, bifunctional anti-adaptor; we are defining the conditions under which AnmK is expressed as a way to investigate its likely biological role. Finally, a long-standing question has been how the cell recovers from stress, in particular from the antiadaptors. We have investigated this process for recovery from phosphate starvation. During this starvation, IraP is induced and stabilizes RpoS. We find that rapid degradation of RpoS is restored rapidly after phosphate is returned to cells, and that this rapid recovery, implying active inactivation of IraP, is dependent on the unstructured C-terminus of IraP. We are investigating the possible roles of cellular proteases in this recovery process, and how it is triggered. Overall, our proteolysis studies continue to provide novel insights into regulatory mechanisms used by bacteria.