The Escherichia coli S2P intramembrane protease RseP regulates ferric citrate uptake by cleaving the sigma factor regulator FecR.

Escherichia coli RseP, a member of the site-2 protease family of intramembrane proteases, is involved in the activation of the σE extracytoplasmic stress response and elimination of signal peptides from the cytoplasmic membrane. However, whether RseP has additional cellular functions is unclear. In this study, we used mass spectrometry-based quantitative proteomic analysis to search for new substrates that might reveal unknown physiological roles for RseP. Our data showed that the levels of several Fec system proteins encoded by the fecABCDE operon (fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with a fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate-dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. A shortened FecR construct was not dependent on RseP for activation, confirming this cleavage step is the essential and sufficient role of RseP. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system.

S2P intramembrane protease RseP degrades small membrane proteins and suppresses the cytotoxicity of intrinsic toxin HokB.

The site2-protease (S2P) family of intramembrane proteases (IMPs) is conserved in all kingdoms of life and cleaves transmembrane proteins within the membrane to regulate and maintain various cellular activities. RseP, an Escherichia coli S2P peptidase, is involved in the regulation of gene expression through the regulated cleavage of the two target membrane proteins (RseA and FecR) and in membrane quality control through the proteolytic elimination of remnant signal peptides. RseP is expected to have additional substrates and to be involved in other cellular processes. Recent studies have shown that cells express small membrane proteins (SMPs; single-spanning membrane proteins of approximately 50-100 amino acid residues) with crucial cellular functions. However, little is known about their metabolism, which affects their functions. This study investigated the possible RseP-catalyzed cleavage of E. coli SMPs based on the apparent similarity of the sizes and structures of SMPs to those of remnant signal peptides. We screened SMPs cleaved by RseP in vivo and in vitro and identified 14 SMPs, including HokB, an endogenous toxin that induces persister formation, as potential substrates. We demonstrated that RseP suppresses the cytotoxicity and biological functions of HokB. The identification of several SMPs as novel potential substrates of RseP provides a clue to a comprehensive understanding of the cellular roles of RseP and other S2P peptidases and highlights a novel aspect of the regulation of SMPs. IMPORTANCE Membrane proteins play an important role in cell activity and survival. Thus, understanding their dynamics, including proteolytic degradation, is crucial. E. coli RseP, an S2P family intramembrane protease, cleaves membrane proteins to regulate gene expression in response to environmental changes and to maintain membrane quality. To identify novel substrates of RseP, we screened small membrane proteins (SMPs), a group of proteins that have recently been shown to have diverse cellular functions, and identified 14 potential substrates. We also showed that RseP suppresses the cytotoxicity of the intrinsic toxin, HokB, an SMP that has been reported to induce persister cell formation, by degrading it. These findings provide new insights into the cellular roles of S2P peptidases and the functional regulation of SMPs.

Cryo-EM structure of the entire FtsH-HflKC AAA protease complex.

The membrane-bound AAA protease FtsH is the key player controlling protein quality in bacteria. Two single-pass membrane proteins, HflK and HflC, interact with FtsH to modulate its proteolytic activity. Here, we present structure of the entire FtsH-HflKC complex, comprising 12 copies of both HflK and HflC, all of which interact reciprocally to form a cage, as well as four FtsH hexamers with periplasmic domains and transmembrane helices enclosed inside the cage and cytoplasmic domains situated at the base of the cage. FtsH K61/D62/S63 in the ß2-ß3 loop in the periplasmic domain directly interact with HflK, contributing to complex formation. Pull-down and in vivo enzymatic activity assays validate the importance of the interacting interface for FtsH-HflKC complex formation. Structural comparison with the substrate-bound human m-AAA protease AFG3L2 offers implications for the HflKC cage in modulating substrate access to FtsH. Together, our findings provide a better understanding of FtsH-type AAA protease holoenzyme assembly and regulation.

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP.

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane ß sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments.

Roles of the Foxj1 and Inv genes in the left-right determination of internal organs in mice.

In Foxj1 knockout mice, half show situs solitus while the other half show situs inversus, which means a random determination of the left-right axis. In contrast, the inv mutant mice show a mirror-image configuration of the internal organs, which means a reversal of the left-right axis. Although these two mutant mice have primary cilia on the nodal cells, their phenotypes are different in laterality determination. We thus made Foxj1/inv double mutant mice and analyzed their phenotype. We found the phenotypes of Foxj1/inv double mutant mice to be more similar to those of the Foxj1 mutant mice than those of the inv mutant mice. We also found right pulmonary isomerism to be a major phenotype of the Foxj1 mutant mice and the Foxj1/inv double mutant mice, which is likely due to the absence of the Pitx2 expression at both lateral plate mesoderms. These results indicate that a random signal of laterality (Foxj1) is dominant over the reversal signal of laterality (Inv).