PDZ domains of RseP are not essential for sequential cleavage of RseA or stress‐induced σ^E activation in vivo

Abstract

SummaryThe Escherichia coli s E extracytoplasmic stress response monitors and responds to folding stress in the cell envelope. A protease cascade directed at RseA, a membrane-spanning anti-s that inhibits s E activity, controls this critical signal-transduction system. Stress cues activate DegS to cleave RseA; a second cleavage by RseP releases RseA from the membrane, enabling its rapid degradation. Stress control of proteolysis requires that RseP cleavage is dependent on DegS cleavage. Recent in vitro and structural studies found that RseP cleavage requires binding of RseP PDZ-C to the newly exposed C-terminal residue (Val148) of RseA, generated by DegS cleavage, explaining dependence. We tested this mechanism in vivo. Neither mutation in the putative PDZ ligand-binding regions nor even deletion of entire RseP PDZ domains had significant effects on RseA cleavage in vivo, and the C-terminal residue of DegSprocessed RseA also little affected RseA cleavage. Indeed, strains with a chromosomal rseP gene deleted for either PDZ domain and strains with a chromosomal rseA V148 mutation grew normally and exhibited almost normal s E activation in response to stress signals. We conclude that recognition of the cleaved amino acid by the RseP PDZ domain is not essential for sequential cleavage of RseA and s E stress response in vivo.

Site-2 protease substrate specificity and coupling in trans by a PDZ-substrate adapter protein

“…Recent structural data suggested that one function of the RseP PDZ domain is to bind the cleaved C terminus of the RseA anti-sigma factor generated by DegS (42), potentially providing a mechanism of coupling. However, recent in vivo examination of this model did not support this mechanism (27). Other recent data indicate that acid can activate RseA cleavage by RseP independent of DegS in S. typhimurium (41) and that LPS biosynthetic intermediates are a second signal for RseA degradation through binding RseB (43).…”

“…The PDZ domain is a widely distributed protein-protein interaction motif that often organizes signaling complexes through binding C-terminal peptides (24). Deletion of the PDZ domain of E. coli RseP causes hyperactivation of the SigE pathway, DegS (S1P), independent cleavage of RseA, and insensitivity of SigE activation to RseB inhibition (20,21,23,27). However, no proteins have been identified that interact with the RseP-PDZ specifically, or S2P-PDZs more broadly, that may mediate these effects.…”

“…This S1P/ S2P coupling necessitates that the S2P remain inactive until the S1P cleavage has occurred, a coupling event that is not well understood. Observations in prokaryotic systems suggest that the PDZ domains of S2Ps participate in this S1P-S2P coupling because this domain is necessary to hold the protease inactive until site one proteolysis occurs (20,21,23,27,41). Recent structural data suggested that one function of the RseP PDZ domain is to bind the cleaved C terminus of the RseA anti-sigma factor generated by DegS (42), potentially providing a mechanism of coupling.…”

Two stress sensor proteins for the expression of sigmaE regulon: DegS and RseB

“…Kanehara et al (2003) suggested that glutamine rich regions in RseA (residues 162-169 and 190-200) suppress RseP-mediated proteolysis, as the removal of the motifs by DegS (the removal of RseA 149-216 ) can induce RseP cleavage. In other studies, residue V148 in RseA exposed by DegS cleavage was required as an RseP activation signal in an in vitro biochemical assay (Li et al, 2009), whereas the residue was not essential for RseA cleavage and sigmaE-dependent transcription in an in vivo assay (Hizukuri and Akiyama, 2012).…”

Function of site-2 proteases in bacteria and bacterial pathogens

“…RseB also controls DegS proteolysis of RseA by binding directly to the periplasmic domain of RseA [10]. DegS cleaved RseA is a substrate for RseP [15–17], although the mechanism of coupling of these two proteolytic events remains controversial [18, 19]. Following RseP cleavage, the RseA/SigE complex is released to the cytoplasm where further degradation of RseA [20] releases SigE to associate with RNA polymerase to activate target promoters.…”