Cells have communication systems that enable them to respond to potentially dangerous changes in their external environment. For example, bacteria have an enzyme called RseP that helps to activate responses to external stresses. This enzyme sits in the membrane that surrounds the cell and cuts a protein called RseA to release a signal molecule into the cell interior. This signal molecule then promotes the expression of particular genes to protect the cell from harm.

Previous studies have identified the ‘active site’ of RseP, which is the region of the enzyme that actually cuts the target protein. However, it is not clear how the enzyme is able to identify and cleave RseA and its other ‘substrate’ proteins. The enzyme also contains a structure called a β-hairpin-like loop that is close to the active site, which is not commonly found in membrane proteins. Here, Akiyama, Mizuno et al. used genetic and biochemical techniques to study the role of this loop structure in the RseP enzyme of the E. coli bacterium.

The experiments show that the loop specifically binds to a section of substrate proteins—called the transmembrane segment—that spans the cell membrane. Several genetic mutations that affected the loop altered the ability of RseP to bind to and cleave substrates. The effect of these mutations in RseP could be suppressed by introducing genetic mutations in substrates that altered the transmembrane segments. Akiyama, Mizuno et al. propose that the β-hairpin-like loop of the RseP enzyme binds the transmembrane segment of a substrate and presents it to the active site.

A previous study showed that another region of RseP called the periplasmic PDZ domains can act as a filter to stop RseP cutting other membrane proteins in error. Akiyama, Mizuno et al.'s findings suggest that the β-hairpin-like loop serves as an additional checkpoint to identify RseA and other proteins that RseP targets. The next step is to carry out further experiments to test this model.

Regulated intramembrane proteolysis (RIP) is a common mechanism for transmembrane signalling and is a part of many important cellular processes in both prokaryotes and eukaryotes (Ha, 2009; Wolfe, 2009; Urban, 2013). Proteases involved in RIP are called intramembrane-cleaving proteases (I-CLiPs) and catalyse the proteolytic cleavage of transmembrane segments (TMs) of target membrane proteins within the lipid bilayer. Escherichia coli RseP belongs to S2P zinc metalloproteinase family of I-CLiPs. It plays a key role in regulating the σ^E extracytoplasmic stress response (Hizukuri et al., 2013; Kroos and Akiyama, 2013), in which the dedicated sigma factor σ^E is activated through the successive cleavage of a single membrane-spanning anti-σ^E RseA in response to cell surface stresses. The first cleavage catalysed by DegS (site-1 cleavage) on the periplasmic side triggers the intramembrane cleavage by RseP (site-2 cleavage) (Alba et al., 2002; Kanehara et al., 2002). Recent studies suggest that in addition to being involved in RseA cleavage, RseP acts in the proteolytic removal of remnant signal peptides from the membrane (Saito et al., 2011). RseP spans the membrane 4 times, with both the termini facing the periplasm. Its central periplasmic region contains tandem PDZ domains (PDZ-N and PDZ-C; Figure 1A) (Kanehara et al., 2001; Kinch et al., 2006; Inaba et al., 2008). The S2P family proteases share a conserved core domain containing 3 TMs. In RseP, these correspond to TM1–TM3 (Figure 1A,B) (Kinch et al., 2006). The first and the third TM contain HExxH and LDG active site motifs, respectively. Disulfide crosslinking and co-immunoprecipitation experiments suggest that TM3 plays a critical role in binding of a substrate TM (Koide et al., 2008). Helix destabilisation of substrate TMs promotes their binding to and cleavage by RseP (Akiyama et al., 2004; Koide et al., 2008).

Figure 1

Download asset Open asset

RseP-catalysed site-2 cleavage of RseA depends on site-1 cleavage (Alba et al., 2002; Kanehara et al., 2002), which results in stress-dependent σ^E activation, because site-1 cleavage is induced by stress signals (Walsh et al., 2003; Kulp and Kuehn, 2011; Lima et al., 2013). Previous studies suggest that RseP PDZ domains are involved in the site-1 cleavage-dependence of the site-2 cleavage by RseP (Kanehara et al., 2003; Bohn et al., 2004; Grigorova et al., 2004; Inaba et al., 2008; Hizukuri and Akiyama, 2012). Although the crystal structure of an S2P homolog (mjS2P) from Methanocaldococcus jannaschii (Feng et al., 2007) has been reported, it does not have a PDZ domain, and the three-dimensional structure of an S2P homolog with PDZ domain(s) is not available. We recently reported the crystal structure of the PDZ tandem of an Aquifex aeolicus RseP homolog. The structure suggests that the 2 PDZ domains create a single pocket-like structure that covers the core membrane domain on the membrane surface (Hizukuri et al., 2014). Structural models and biochemical and genetic results suggest that the PDZ tandem acts as a size-exclusion filter to allow only the periplasmically truncated form of RseA to enter the recessed active site in the membrane domain of RseP (Kroos and Akiyama, 2013; Hizukuri et al., 2014). However, mechanisms underlying substrate proteolysis by RseP remains elusive. It is unclear whether the size-exclusion function of the PDZ tandem is sufficient to discriminate between substrates and non-substrates and how a substrate TM, which assumes an α-helical conformation that needs to be unwound for proteolysis, is recognised and presented to the proteolytic active site. The mechanism of substrate discrimination and specific cleavage in RIP is one of the major problems to be solved (Langosch et al., 2015). Detailed knowledge of mechanisms underlying substrate discrimination and cleavage would help in controlling the cleavage of membrane proteins by I-CLiPs including S2P proteases.

The structure of mjS2P indicates that it contains a region, located close to the active site, that is looped into the membrane domain from the cytoplasmic side (Figure 1B,C). This membrane-reentrant loop is unique because it is in an extended or β-strand conformation, whereas a membrane-embedded polypeptide is generally in an α-helical conformation. Therefore, we have named this region membrane-reentrant β-loop (MRE β-loop). The MRE β-loop is conserved in proteins belonging to groups I and III of S2P subfamilies, including RseP (group I), Bacillus subtilis SpoIVFB (group III) and mjS2P (group III; Figure 1D) (Ha, 2009; Kroos and Akiyama, 2013; Zhang et al., 2013). A recent study showed that the MRE β-loop of SpoIVFB can be crosslinked with a substrate through disulfide bonds, suggesting its possible involvement in substrate interaction (Zhang et al., 2013). However, functional roles of the MRE β-loop remain largely unknown. Here, we analysed the function of the RseP MRE β-loop and found that it could directly bind to and promotes selective cleavage of RseP substrates. Therefore, we propose that the MRE β-loop stabilises substrate TMs in an extended conformation and presents them to the proteolytic active site.

RseP is one of the most extensively characterised proteins among the S2P family of I-CLiPs. However, detailed mechanisms underlying its substrate recognition and cleavage are not completely understood. We focused on the MRE β-loop, a conserved intramembrane β-hairpin-like structure, and investigated its role in substrate proteolysis by RseP. Deletion or Pro substitution in the MRE β-loop impaired substrate cleavage, indicating its critical role in the proteolytic function of RseP. Crosslinking and co-immunoprecipitation experiments showed that the MRE β-loop was directly involved in substrate binding. Several Pro substitutions differentially affected RseP-catalysed proteolysis of substrates. Helix-destabilising mutations in substrate TMs suppressed the defects caused by mutations in the MRE β-loop in an allele-specific manner. These results collectively suggest that the MRE β-loop specifically recognises substrate TMs.

The proximal part of the MRE β-loop has a short β-strand (Ile⁷²-Leu-Leu) in the reported crystal structure of mjS2P (Feng et al., 2007). However, no secondary structure is assigned for the distal part of the MRE β-loop. Analysis of the secondary structure with STRIDE (Frishman and Argos, 1995) suggested that Val⁸⁰-Ala-Met in the distal part forms a β-strand (Figure 1B–D). This region is in an extended conformation with the side chains facing the opposite sides in an alternate manner (as expected for a β-strand) suggesting that this region forms a β-strand. Sequence-based prediction suggested that the MRE β-loops of RseP and SpoIVFB also have β-strands in the corresponding regions (Figure 1D), suggesting that the MRE β-loop commonly assumes a β-hairpin-like structure.

Cys-scanning mutagenesis suggest that no specific residue in the MRE β-loop is essential for the proteolytic function of RseP because the Cys substitutions had little or only slight effect on the substrate cleavage. In contrast, substrate cleavage was severely impaired by Pro substitutions. These observations suggest that a higher-order structure of the MRE β-loop is important for the proteolytic function of RseP. Interestingly, some Pro substitutions in the MRE β-loop resulted in the differential cleavage of the 3 model substrates. Pulse-chase experiments showed that RseP(G68P)-HM significantly cleaved LY1 but only slightly cleaved RseA TM and YqfG TM. In contrast, RseP(K71P)-HM efficiently cleaved RseA TM and LY1 but only slightly cleaved YqfG TM. These 3 substrates were rapidly cleaved by wild-type RseP-HM. Therefore, the effects of MRE β-loop mutations cannot be simply explained by the intrinsic susceptibility of these substrates to RseP. In addition, some mutations in YqfG TM suppressed G67P and K71P mutations of the RseP MRE β-loop in an allele-specific manner. The I8P mutation improved the proteolysis by RseP(K71P)-HM but not by RseP(G67P)-HM, whereas the L12P mutation improved the proteolysis by RseP(G67P)-HM compared with that of RseP(K71P)-HM. Similarly the F21P, but not F20P, mutation in the LacY TM1 region (LY1) partially suppressed the V70P mutation of the RseP MRE β-loop. These results suggest that the MRE β-loop specifically recognises substrate TMs.

Direct evidence of MRE β-loop–substrate interaction was obtained by co-immunoprecipitation and crosslinking experiments. Pro substitutions in the MRE β-loop markedly decreased the co-immunoprecipitation of RseP-HM with HA-MBP-RseA after membrane solubilisation, suggesting that the integrity of the MRE β-loop is required for stable RseP–substrate interaction. Disulfide- and photo-crosslinking experiments showed that residues at positions 69 and 71 of the MRE β-loop contact with substrates. Cys residues at these positions disulfide-bonded with Cys residues on either side of the scissile bond in RseA. A similar result was reported recently for SpoIVFB and its substrate Pro-σ^K (Zhang et al., 2013). A Cys residue in place of Val-70 in the MRE β-loop of SpoIVFB formed disulfide bonds with Cys residues at several positions around the cleavage site in Pro-σ^K, suggesting that the MRE β-loop of SpoIVFB directly interacts with Pro-σ^K. However, the role of the MRE β-loop in SpoIVFB function has not been investigated experimentally. Although RseP also has a Val residue at the position corresponding to Val-70 in SpoIVFB (Figure 1D), we did not observe any substrate crosslinking at this position. This might be due to differences in the mode of enzyme–substrate interaction between RseP and SpoIVFB. Indeed, the substrates of these enzymes show some differences. In contrast to RseA, which is a single-spanning membrane protein with type II orientation, Pro-σ^K is suggested to be peripherally associated with the membrane (Zhou et al., 2013). In addition, although helix-destabilising residues in substrate TMs are important for their efficient cleavage by RseP, these residues are not required for the cleavage of Pro-σ^K by SpoIVFB (Zhou et al., 2013). Thus, the role of the MRE β-loop in substrate binding and cleavage might slightly differ between RseP and SpoIVFB.

The N389L mutation in RseP TM3, which was previously shown to weaken the RseP-substrate interaction (Koide et al., 2008), inhibited the in vivo photo-crosslinking of the MRE β-loop with RseA, suggesting that the observed crosslinking reflected a functional RseP–substrate interaction. The TM4 (corresponding to RseP TM3) of mjS2P that contains one of the active site residues (corresponding to Asp-402 in RseP TM3) can be located in the vicinity of the proteolytic active site and the MRE β-loop (Feng et al., 2007). We previously showed that Cys introduced at the positions of Pro-397 and Pro-399 in RseP TM3 can form a disulfide bond with Cys at multiple positions of RseA TM. In mjS2P, the residues corresponding to Pro-397 and Pro-399 of RseP reside in a loop-like structure. The region containing residues 397 and 399 might be flexible and act with the MRE β-loop in stable binding of a substrate.

An α-helix is generally not amenable to proteolysis and unwinds to an extended structure to undergo cleavage (Wolfe, 2009). Many zinc metalloproteinases have a β-strand (edge strand) close to their proteolytic active site that binds to substrates in an extended conformation through β-strand addition and presents them to catalytic residues (Stocker and Bode, 1995; Langklotz et al., 2012). Our results suggest that the MRE β-loop stabilises the extended conformation of a substrate by directly binding to it through β-strand addition, thus promoting substrate cleavage in a manner similar to the edge strand (Figure 7). Crosslinking at alternate positions in the Tyr⁶⁹-Val-Lys region supports the idea that this region is in a β-strand conformation during its interaction with a substrate. According to this model, mutations that disrupt the secondary structure of the distal part of the MRE β-loop would compromise its interaction with a substrate. Introduction of a Pro residue will disturb substrate accommodation through β-strand addition or interstrand interaction because Pro residues cannot serve as hydrogen bond donors because of the absence of the amide proton. Moreover, if the compromised interaction of the MRE β-loop with a substrate prevents efficient conformational conversion of the substrate TM and decreases its cleavage efficiency, destabilisation of the α-helical structure of the substrate TM might improve its cleavage by RseP MRE β-loop mutants, which is consistent with our results. A recent structural study of E. coli GlpG that belongs to the rhomboid family of I-CLiPs revealed that the P1–P4 region of a substrate forms a β-sheet with the GlpG loop 3 located near the active site (Zoll et al., 2014). Mutations in loop 3 severely compromise the GlpG activity, suggesting the interaction between the substrate and loop 3 is functionally important (Baker and Urban, 2012). Although the loop 3 of GlpG and the MRE β-loop of RseP differ in that the former is located near the periplasmic surface of the membrane and forms a parallel β-sheet with a substrate whereas the latter is located near the cytoplasmic surface and forms a anti-parallel β-sheet, they might have a similar role in stabilising an extended substrate structure and promoting its cleavage.

Figure 7

Download asset Open asset

Differential effects of Pro substitutions in the MRE β-loop on the cleavage of the model substrates and allele-specific suppression of these mutations by helix-destabilising mutations in substrate TMs suggest that the MRE β-loop–substrate interaction occurs with some specificity. MRE β-loop-assisted cleavage of a substrate may be affected by helix stability and amino acid sequence of substrate TMs. We recently suggested that the periplasmic PDZ tandem of RseP acts as a size-exclusion filter to prevent the cleavage of substrates with bulky periplasmic domains (Figure 7A) (Hizukuri et al., 2014). In this study, we observed that RseP did not cleave YoaJ, a type II membrane protein with a very small periplasmic domain, suggesting the presence of additional mechanism(s) for substrate discrimination. In vivo photo-crosslinking experiments suggest that the MRE β-loop interacts with RseA after site 1 cleavage (Figure 7B) and that it acts as the second checkpoint for membrane proteins that passed the first check by the PDZ filter and contributes to their selective proteolysis. It should be noted that mjS2P does not have PDZ domains. Thus, the MRE β-loop could play a central role in cleaving specific substrates in this enzyme.

Although the MRE β-loop may not interact with intact RseA, our previous chemical crosslinking and co-immunoprecipitation experiments showed that RseP directly interacted with intact RseA in the membrane (Kanehara et al., 2003), suggesting that RseP has a separate binding site (exosite) for intact RseA. Because the PDZ filter excludes intact RseA from the intramolecular active site, the presumed exosite may exist on the external surface of the enzyme. Other families of I-CLiPs such as rhomboid and γ-secretase have also been suggested to have an exosite (Kornilova et al., 2005; Strisovsky et al., 2009; Watanabe et al., 2010; Arutyunova et al., 2014). A systematic in vivo crosslinking approach would help in identifying binding sites for intact RseA. This along with the structural analysis of RseP, especially in complex with a substrate, would be essential for verifying our model and for understanding molecular mechanisms underlying substrate recognition and intramembrane proteolysis by RseP. Further, we would like to study the physiological significance of RseP-catalysed cleavage of YqfG and identify additional substrates of RseP to elucidate all the cellular roles of RseP.

1. 1. Akiyama Y
   2. Kanehara K
   3. Ito K

   (2004) RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences

   The EMBO Journal 23:4434–4442.

   https://doi.org/10.1038/sj.emboj.7600449

   • Google Scholar
2. 1. Alba BM
   2. Leeds JA
   3. Onufryk C
   4. Lu CZ
   5. Gross CA

   (2002) DegS and YaeL participate sequentially in the cleavage of RseA to activate the σ^E-dependent extracytoplasmic stress response

   Genes & Development 16:2156–2168.

   https://doi.org/10.1101/gad.1008902

   • Google Scholar
3. 1. Alix E
   2. Blanc-Potard AB

   (2009) Hydrophobic peptides: novel regulators within bacterial membrane

   Molecular Microbiology 72:5–11.

   https://doi.org/10.1111/j.1365-2958.2009.06626.x

   • Google Scholar
4. 1. Arutyunova E
   2. Panwar P
   3. Skiba PM
   4. Gale N
   5. Mak MW
   6. Lemieux MJ

   (2014) Allosteric regulation of rhomboid intramembrane proteolysis

   The EMBO Journal 33:1869–1881.

   https://doi.org/10.15252/embj.201488149

   • Google Scholar
5. 1. Baba T
   2. Ara T
   3. Hasegawa M
   4. Takai Y
   5. Okumura Y
   6. Baba M
   7. Datsenko KA
   8. Tomita M
   9. Wanner BL
   10. Mori H

   (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection

   Molecular Systems Biology 2:2006.0008.

   https://doi.org/10.1038/msb4100050

   • Google Scholar
6. 1. Baker RP
   2. Urban S

   (2012) Architectural and thermodynamic principles underlying intramembrane protease function

   Nature Chemical Biology 8:759–768.

   https://doi.org/10.1038/nchembio.1021

   • Google Scholar
7. 1. Bohn C
   2. Collier J
   3. Bouloc P

   (2004) Dispensable PDZ domain of Escherichia coli YaeL essential protease

   Molecular Microbiology 52:427–435.

   https://doi.org/10.1111/j.1365-2958.2004.03985.x

   • Google Scholar
8. 1. Datsenko KA
   2. Wanner BL

   (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

   Proceedings of the National Academy of Sciences of USA 97:6640–6645.

   https://doi.org/10.1073/pnas.120163297

   • Google Scholar
9. 1. Douchin V
   2. Bohn C
   3. Bouloc P

   (2006) Down-regulation of porins by a small RNA bypasses the essentiality of the regulated intramembrane proteolysis protease RseP in Escherichia coli

   The Journal of Biological Chemistry 281:12253–12259.

   https://doi.org/10.1074/jbc.M600819200

   • Google Scholar
10. 1. Feng L
    2. Yan H
    3. Wu Z
    4. Yan N
    5. Wang Z
    6. Jeffrey PD
    7. Shi Y

    (2007) Structure of a site-2 protease family intramembrane metalloprotease

    Science 318:1608–1612.

    https://doi.org/10.1126/science.1150755

    • Google Scholar
11. 1. Flynn JM
    2. Levchenko I
    3. Sauer RT
    4. Baker TA

    (2004) Modulating substrate choice: the SspB adaptor delivers a regulator of the extracytoplasmic-stress response to the AAA+ protease ClpXP for degradation

    Genes & Development 18:2292–2301.

    https://doi.org/10.1101/gad.1240104

    • Google Scholar
12. 1. Fontaine F
    2. Fuchs RT
    3. Storz G

    (2011) Membrane localization of small proteins in Escherichia coli

    The Journal of Biological Chemistry 286:32464–32474.

    https://doi.org/10.1074/jbc.M111.245696

    • Google Scholar
13. 1. Frishman D
    2. Argos P

    (1995) Knowledge-based protein secondary structure assignment

    Proteins 23:566–579.

    https://doi.org/10.1002/prot.340230412

    • Google Scholar
14. 1. Grigorova IL
    2. Chaba R
    3. Zhong HJ
    4. Alba BM
    5. Rhodius V
    6. Herman C
    7. Gross CA

    (2004) Fine-tuning of the Escherichia coli σ^E envelope stress response relies on multiple mechanisms to inhibit signal-independent proteolysis of the transmembrane anti-sigma factor, RseA

    Genes & Development 18:2686–2697.

    https://doi.org/10.1101/gad.1238604

    • Google Scholar
15. 1. Ha Y

    (2009) Structure and mechanism of intramembrane protease

    Seminars in Cell & Developmental Biology 20:240–250.

    https://doi.org/10.1016/j.semcdb.2008.11.006

    • Google Scholar
16. 1. Hemm MR
    2. Paul BJ
    3. Schneider TD
    4. Storz G
    5. Rudd KE

    (2008) Small membrane proteins found by comparative genomics and ribosome binding site models

    Molecular Microbiology 70:1487–1501.

    https://doi.org/10.1111/j.1365-2958.2008.06495.x

    • Google Scholar
17. 1. Hizukuri Y
    2. Akiyama Y

    (2012) PDZ domains of RseP are not essential for sequential cleavage of RseA or stress-induced σ^E activation in vivo

    Molecular Microbiology 86:1232–1245.

    https://doi.org/10.1111/mmi.12053

    • Google Scholar
18. Book

    1. Hizukuri Y
    2. Ito K
    3. Akiyama Y

    (2013)

    RseP peptidase

    In: Rawlings ND, Salvesen G, editors. Handbook of proteolytic enzymes (3rd editon). Elsevier Ltd, Amsterdam. pp. 1546–1550.

    • Google Scholar
19. 1. Hizukuri Y
    2. Oda T
    3. Tabata S
    4. Tamura-Kawakami K
    5. Oi R
    6. Sato M
    7. Takagi J
    8. Akiyama Y
    9. Nogi T

    (2014) A structure-based model of substrate discrimination by a noncanonical PDZ tandem in the intramembrane-cleaving protease RseP

    Structure 22:326–336.

    https://doi.org/10.1016/j.str.2013.12.003

    • Google Scholar
20. 1. Inaba K
    2. Suzuki M
    3. Maegawa K
    4. Akiyama S
    5. Ito K
    6. Akiyama Y

    (2008) A pair of circularly permutated PDZ domains control RseP, the S2P family intramembrane protease of Escherichia coli

    The Journal of Biological Chemistry 283:35042–35052.

    https://doi.org/10.1074/jbc.M806603200

    • Google Scholar
21. 1. Ito K
    2. Akiyama Y

    (1991) In vivo analysis of integration of membrane proteins in Escherichia coli

    Molecular Microbiology 5:2243–2253.

    https://doi.org/10.1111/j.1365-2958.1991.tb02154.x

    • Google Scholar
22. 1. Kanehara K
    2. Akiyama Y
    3. Ito K

    (2001) Characterization of the yaeL gene product and its S2P-protease motifs in Escherichia coli

    Gene 281:71–79.

    https://doi.org/10.1016/S0378-1119(01)00823-X

    • Google Scholar
23. 1. Kanehara K
    2. Ito K
    3. Akiyama Y

    (2002) YaeL (EcfE) activates the σ^E pathway of stress response through a site-2 cleavage of anti-σ^E, RseA

    Genes & Development 16:2147–2155.

    https://doi.org/10.1101/gad.1002302

    • Google Scholar
24. 1. Kanehara K
    2. Ito K
    3. Akiyama Y

    (2003) YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA

    The EMBO Journal 22:6389–6398.

    https://doi.org/10.1093/emboj/cdg602

    • Google Scholar
25. 1. Kihara A
    2. Akiyama Y
    3. Ito K

    (2001) Revisiting the lysogenization control of bacteriophage λ. Identification and characterization of a new host component, HflD

    The Journal of Biological Chemistry 276:13695–13700.

    https://doi.org/10.1074/jbc.M011699200

    • Google Scholar
26. 1. Kinch LN
    2. Ginalski K
    3. Grishin NV

    (2006) Site-2 protease regulated intramembrane proteolysis: sequence homologs suggest an ancient signaling cascade

    Protein Science 15:84–93.

    https://doi.org/10.1110/ps.051766506

    • Google Scholar
27. 1. Koide K
    2. Ito K
    3. Akiyama Y

    (2008) Substrate recognition and binding by RseP, an Escherichia coli intramembrane protease

    The Journal of Biological Chemistry 283:9562–9570.

    https://doi.org/10.1074/jbc.M709984200

    • Google Scholar
28. 1. Kornilova AY
    2. Bihel F
    3. Das C
    4. Wolfe MS

    (2005) The initial substrate-binding site of γ-secretase is located on presenilin near the active site

    Proceedings of the National Academy of Sciences of USA 102:3230–3235.

    https://doi.org/10.1073/pnas.0407640102

    • Google Scholar
29. 1. Krogh A
    2. Larsson B
    3. von Heijne G
    4. Sonnhammer EL

    (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes

    Journal of Molecular Biology 305:567–580.

    https://doi.org/10.1006/jmbi.2000.4315

    • Google Scholar
30. 1. Kroos L
    2. Akiyama Y

    (2013) Biochemical and structural insights into intramembrane metalloprotease mechanisms

    Biochimica et Biophysica Acta 1828:2873–2885.

    https://doi.org/10.1016/j.bbamem.2013.03.032

    • Google Scholar
31. 1. Kulp A
    2. Kuehn MJ

    (2011) Recognition of β-strand motifs by RseB is required for σ^E activity in Escherichia coli

    Journal of Bacteriology 193:6179–6186.

    https://doi.org/10.1128/JB.05657-11

    • Google Scholar
32. 1. Langklotz S
    2. Baumann U
    3. Narberhaus F

    (2012) Structure and function of the bacterial AAA protease FtsH

    Biochimica et Biophysica Acta 1823:40–48.

    https://doi.org/10.1016/j.bbamcr.2011.08.015

    • Google Scholar
33. 1. Langosch D
    2. Scharnagl C
    3. Steiner H
    4. Lemberg MK

    (2015) Understanding intramembrane proteolysis: from protein dynamics to reaction kinetics

    Trends in Biochemical Sciences 40:318–327.

    https://doi.org/10.1016/j.tibs.2015.04.001

    • Google Scholar
34. 1. Lima S
    2. Guo MS
    3. Chaba R
    4. Gross CA
    5. Sauer RT

    (2013) Dual molecular signals mediate the bacterial response to outer-membrane stress

    Science 340:837–841.

    https://doi.org/10.1126/science.1235358

    • Google Scholar
35. Book

    1. Miller JH

    (1972)

    Experiments in molecular genetics

    Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

    • Google Scholar
36. 1. Narita S
    2. Masui C
    3. Suzuki T
    4. Dohmae N
    5. Akiyama Y

    (2013) Protease homolog BepA (YfgC) promotes assembly and degradation of β-barrel membrane proteins in Escherichia coli

    Proceedings of the National Academy of Sciences of USA 110:E3612–E3621.

    https://doi.org/10.1073/pnas.1312012110

    • Google Scholar
37. 1. Saito A
    2. Hizukuri Y
    3. Matsuo E
    4. Chiba S
    5. Mori H
    6. Nishimura O
    7. Ito K
    8. Akiyama Y

    (2011) Post-liberation cleavage of signal peptides is catalyzed by the site-2 protease (S2P) in bacteria

    Proceedings of the National Academy of Sciences of USA 108:13740–13745.

    https://doi.org/10.1073/pnas.1108376108

    • Google Scholar
38. 1. Schrödinger LLC

    (2010) The PyMOL molecular graphics system, version 1.3r1

    The PyMOL molecular graphics system, version 1.3r1, https://www.pymol.org/.

    https://www.pymol.org/

    • Google Scholar
39. 1. Stocker W
    2. Bode W

    (1995) Structural features of a superfamily of zinc-endopeptidases: the metzincins

    Current Opinion in Structural Biology 5:383–390.

    https://doi.org/10.1016/0959-440X(95)80101-4

    • Google Scholar
40. 1. Strisovsky K
    2. Sharpe HJ
    3. Freeman M

    (2009) Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates

    Molecular Cell 36:1048–1059.

    https://doi.org/10.1016/j.molcel.2009.11.006

    • Google Scholar
41. 1. Urban S

    (2013) Mechanisms and cellular functions of intramembrane proteases

    Biochimica et Biophysica Acta 1828:2797–2800.

    https://doi.org/10.1016/j.bbamem.2013.07.001

    • Google Scholar
42. 1. Walsh NP
    2. Alba BM
    3. Bose B
    4. Gross CA
    5. Sauer RT

    (2003) OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain

    Cell 113:61–71.

    https://doi.org/10.1016/S0092-8674(03)00203-4

    • Google Scholar
43. 1. Watanabe N
    2. Takagi S
    3. Tominaga A
    4. Tomita T
    5. Iwatsubo T

    (2010) Functional analysis of the transmembrane domains of presenilin 1: participation of transmembrane domains 2 and 6 in the formation of initial substrate-binding site of γ-secretase

    The Journal of Biological Chemistry 285:19738–19746.

    https://doi.org/10.1074/jbc.M110.101287

    • Google Scholar
44. 1. Wolfe MS

    (2009) Intramembrane proteolysis

    Chemical Reviews 109:1599–1612.

    https://doi.org/10.1021/cr8004197

    • Google Scholar
45. 1. Young TS
    2. Ahmad I
    3. Yin JA
    4. Schultz PG

    (2010) An enhanced system for unnatural amino acid mutagenesis in E. coli

    Journal of Molecular Biology 395:361–374.

    https://doi.org/10.1016/j.jmb.2009.10.030

    • Google Scholar
46. 1. Zhang Y
    2. Luethy PM
    3. Zhou R
    4. Kroos L

    (2013) Residues in conserved loops of intramembrane metalloprotease SpoIVFB interact with residues near the cleavage site in pro-σ^K

    Journal of Bacteriology 195:4936–4946.

    https://doi.org/10.1128/JB.00807-13

    • Google Scholar
47. 1. Zhou R
    2. Chen K
    3. Xiang X
    4. Gu L
    5. Kroos L

    (2013) Features of Pro-σ^K important for cleavage by SpoIVFB, an intramembrane metalloprotease

    Journal of Bacteriology 195:2793–2806.

    https://doi.org/10.1128/JB.00229-13

    • Google Scholar
48. 1. Zoll S
    2. Stanchev S
    3. Began J
    4. Skerle J
    5. Lepsik M
    6. Peclinovska L
    7. Majer P
    8. Strisovsky K

    (2014) Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures

    The EMBO Journal 33:2408–2421.

    https://doi.org/10.15252/embj.201489367

    • Google Scholar

Author details

1. Koichiro Akiyama

   Institute for Virus Research, Kyoto University, Kyoto, Japan

   Contribution

   KA, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Shinya Mizuno

   Competing interests

   The authors declare that no competing interests exist.

2. Shinya Mizuno

   Institute for Virus Research, Kyoto University, Kyoto, Japan

   Contribution

   SM, Conception and design, Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   Koichiro Akiyama

   Competing interests

   The authors declare that no competing interests exist.

3. Yohei Hizukuri

   Institute for Virus Research, Kyoto University, Kyoto, Japan

   Contribution

   YH, Conception and design, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Hiroyuki Mori

   Institute for Virus Research, Kyoto University, Kyoto, Japan

   Contribution

   HM, Conception and design, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. Terukazu Nogi

   Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan

   Contribution

   TN, Conception and design, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

6. Yoshinori Akiyama

   Institute for Virus Research, Kyoto University, Kyoto, Japan

   Contribution

   YA, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   yakiyama@virus.kyoto-u.ac.jp

   Competing interests

   The authors declare that no competing interests exist.

• 1,339

  views

• 285

  downloads

• 39

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.