Involvement of a Membrane-Bound Amphiphilic Helix in Substrate Discrimination and Binding by an Escherichia coli S2P Peptidase RseP

Miyake, Tatsuya; Hizukuri, Yohei; Akiyama, Yoshinori

doi:10.3389/fmicb.2020.607381

Cited by 9 publications

(26 citation statements)

References 59 publications

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“…Monoclonal anti-FLAG M2 antibody (MilliporeSigma), Rabbit polyclonal anti-RseP antibody ( 82 ), and anti-SecB antibody ( 26 ) were used for immunoblotting. Monoclonal anti-FLAG M2 Affinity Gel (MilliporeSigma) was used for Immunoprecipitation in pulse-chase assay.…”

Section: Methodsmentioning

confidence: 99%

“…They include the production of sex pheromones in Enterococcus faecalis ( 15 ), sporulation in Bacillus subtilis ( 16 ), acid response in Salmonella enterica ( 17 ), mucoid conversion and alginate overproduction in Pseudomonas aeruginosa ( 18 ), production of cholera toxin in Vibrio cholerae ( 19 ), and iron acquisition in Bordetella bronchiseptica and P. aeruginosa ( 20 , 21 ), RseP spans the membrane four times with both of its N terminus and C terminus facing the periplasmic space. The residues of the first and third TM segments constitute the intramembrane proteolytic active site ( 22 , 23 ), and the central periplasmic region between the second and the third TM segments contains tandemly arranged two PDZ domains (PDZ tandem) ( 24 , 25 ) and an amphiphilic helix that is presumably involved in the proper positioning of the PDZ tandem and a substrate ( 26 ). RseP and other S2P proteases generally cleave a single-spanning membrane protein with type II (N IN -C OUT ) topology.…”

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confidence: 99%

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

Yokoyama

Niinae

Tsumagari

et al. 2021

Journal of Biological Chemistry

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Escherichia coli RseP, a member of the site-2 protease family of intramembrane proteases, is involved in the activation of the σ E 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.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Yokoyama

Niinae

Tsumagari

et al. 2021

Journal of Biological Chemistry

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“…Monoclonal anti-FLAG M2 antibody (MilliporeSigma), Rabbit polyclonal anti-RseP antibody (83) and anti-SecB antibody (26) were used for immunoblotting. Monoclonal anti-FLAG M2 Affinity Gel (MilliporeSigma) was used for Immunoprecipitation in pulse-chase assay.…”

Section: Methodsmentioning

confidence: 99%

“…RseP spans the membrane 4 times with both of its N- and C-termini facing the periplasmic space. The residues of the first and third transmembrane segments constitute the intramembrane proteolytic active site (22, 23), and the central periplasmic region between the second and the third transmembrane segments contains tandemly-arranged two PDZ domains (PDZ tandem) (24, 25) and an amphiphilic helix that is presumably involved in the proper positioning of the PDZ tandem and a substrate (26). RseP and other S2P proteases generally cleave a single-spanning membrane protein with type II (NIN-COUT) topology.…”

Section: Introductionmentioning

confidence: 99%

Escherichia coliS2P family intramembrane protease RseP is engaged in the regulated sequential cleavages of FecR in the ferric citrate signaling

Yokoyama

Niinae

Tsumagari

et al. 2021

Preprint

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Escherichia coli RseP, a member of the S2P family of intramembrane proteases, is involved in the activation of the σE extracytoplasmic stress response and elimination of remnant signal peptides. However, whether RseP has additional cellular functions is unclear. In this study, we attempted to identify new RseP substrates to explore still unknown physiological roles of this protease. Our mass spectrometry-based quantitative proteomic analysis revealed 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 the 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. Ferric citrate signal-dependent generation of this cleavage product is the essential and sufficient role of RseP in the transcriptional activation of the fec operon. 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.

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“…Immunoblots with anti-HA, anti-His or anti-SecB antibodies were visualized using a Lumino LAS 4000 mini image analyzer (Cytiva) with ECL Prime Western Blotting Detection Reagents (Cytiva). Rabbit polyclonal anti-HA [HA-probe (Y-11), Santa Cruz Biotechnology] and anti-SecB (Miyake et al, 2020) antibodies were used for immunoblotting. For the detection of His-tagged proteins, anti-His antibodies from the Penta-His HRP Conjugate Kit (Qiagen) were used.…”

Section: In Vivo Cleavage Assay Of Aarsep and Its Mutantsmentioning

confidence: 99%

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

Tamura-Sakaguchi

Aruga

Hirose

et al. 2021

Acta Cryst Sect D Struct Biol

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Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

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Involvement of a Membrane-Bound Amphiphilic Helix in Substrate Discrimination and Binding by an Escherichia coli S2P Peptidase RseP

Cited by 9 publications

References 59 publications

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

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

Escherichia coliS2P family intramembrane protease RseP is engaged in the regulated sequential cleavages of FecR in the ferric citrate signaling

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

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