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

2004; Springer Nature; Volume: 23; Issue: 22 Linguagem: Inglês

10.1038/sj.emboj.7600449

1460-2075

Yoshinori Akiyama, Kazue Kanehara, Koreaki Ito,

Bacterial Genetics and Biotechnology

Article21 October 2004free access RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences Yoshinori Akiyama Corresponding Author Yoshinori Akiyama Institute for Virus Research, Kyoto University, Kyoto, Japan Search for more papers by this author Kazue Kanehara Kazue Kanehara Search for more papers by this author Koreaki Ito Koreaki Ito Search for more papers by this author Yoshinori Akiyama Corresponding Author Yoshinori Akiyama Institute for Virus Research, Kyoto University, Kyoto, Japan Search for more papers by this author Kazue Kanehara Kazue Kanehara Search for more papers by this author Koreaki Ito Koreaki Ito Search for more papers by this author Author Information Yoshinori Akiyama 1, Kazue Kanehara and Koreaki Ito 1Institute for Virus Research, Kyoto University, Kyoto, Japan *Corresponding author. Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan. Tel.: +81 75 751 4040; Fax: +81 75 761 5626 or +81 75 771 5699; E-mail: [email protected] The EMBO Journal (2004)23:4434-4442https://doi.org/10.1038/sj.emboj.7600449 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Escherichia coli RseP (formerly YaeL) is believed to function as a 'regulated intramembrane proteolysis' (RIP) protease that introduces the second cleavage into anti-σE protein RseA at a position within or close to the transmembrane segment. However, neither its enzymatic activity nor the substrate cleavage position has been established. Here, we show that RseP-dependent cleavage indeed occurs within predicted transmembrane sequences of membrane proteins in vivo. Moreover, RseP catalyzed the same specificity proteolysis in an in vitro reaction system using purified components. Our in vivo and in vitro results show that RseP can cleave transmembrane sequences of some model membrane proteins that are unrelated to RseA, provided that the transmembrane region contains residues of low helical propensity. These results show that RseP has potential ability to cut a broad range of membrane protein sequences. Intriguingly, it is nevertheless recruited to the σE stress-response cascade as a specific player of RIP. Introduction Proteolytic processes have increasing importance in regulation of cellular functions. Not only cytosolic proteins but proteins integral to biological membranes receive proteolysis. There are at least two general categories in the proteolysis of membrane proteins. One is their rapid elimination when they have failed proper assembly or folding, and the other is their functional conversion by specific cleavages. The latter reactions can be accompanied by liberation of a functional domain from the membrane, thus allowing its altered subcellular localization. In fact, this category of proteolytic process is called regulated intramembrane proteolysis (RIP) and known to be widespread in organisms, from prokaryotes to eukaryotes (Brown et al, 2000; Weihofen and Martoglio, 2003; Wolfe and Kopan, 2004). It occurs in response to a specific regulatory signal and provides cells with a means to undergo a unidirectional change to a new physiological state. Depending on the topological arrangement of the site of signal reception and the output domain, this regulation can be transmembrane (TM). Proteases responsible for RIP are all multispanning membrane proteins, which can be classified into four families: Presenilin, S2P protease, signal peptide peptidase (SPP) and Rhomboid protease (Brown et al, 2000; Weihofen and Martoglio, 2003; Wolfe and Kopan, 2004). Among RIP proteases, S2P is involved in regulation of sterol metabolism and unfolded protein response through its activity to cleave SREBP (Rawson et al, 1997) and ATF6 (Ye et al, 2000b), respectively. YaeL, now renamed RseP (regulator of sigma E, protease), is an Escherichia coli ortholog of the S2P protease (Brown et al, 2000; Kanehara et al, 2001). It is a membrane protein having zinc metalloprotease active-site motifs and four TM segments (Kanehara et al, 2001) and essential for cell viability through its ability to activate the σE pathway of extracytoplasmic stress responses (Alba et al, 2002; Kanehara et al, 2002). σE is an alternative sigma factor responsible for transcription of genes for cell surface proteins involved in biogenesis and quality control of the envelope structure (Ades, 2004; Alba and Gross, 2004). Under normal growth conditions, σE is kept inactive by its interaction with RseA, a single-spanning membrane protein having N-terminal anti-σE domain exposed to the cytosol (De Las Peñas et al, 1997; Missiakas et al, 1997). Accumulation of abnormal envelope proteins activates DegS, a membrane-bound serine protease with a periplasmic active site, to introduce 'site-1' cleavage into the RseA periplasmic region (Walsh et al, 2003). The DegS-cleaved form of RseA (RseA(ΔP)) is then subject to RseP-dependent 'site-2' cleavage at a more cytoplasmically proximal site (Alba et al, 2002; Kanehara et al, 2002). Thus, the overall output of the extracytoplasmic stress signal is degradation of RseA and consequent activation of σE. Degradation of RseA, a key mechanism of the σE activation, is under strict regulation by the cell surface status (Ades, 2004; Alba and Gross, 2004). Recent studies have shown that two PDZ-like domains, one in DegS and the other in RseP, play critical regulatory roles in this stress-response mechanism (Kanehara et al, 2003; Walsh et al, 2003; Bohn et al, 2004; Wilken et al, 2004). The PDZ domain of DegS binds to the stress-dependently exposed outer membrane signature sequence and then transforms DegS into an enzymatically active protease (Wilken et al, 2004). The periplasmic PDZ domain of RseP, in conjunction with the Gln-rich regions in the periplasmic domain of RseA, prevents uncontrolled cleavage of RseA without its DegS-mediated first cleavage (Kanehara et al, 2003; Bohn et al, 2004). The question of how these periplasmic elements negatively control the enzymatic action against the intact RseA remains to be addressed. Although RseP is believed to be a protease that introduces a single cut into RseA at an intramembrane or cytosolic site, its protease activity has not been demonstrated biochemically, and even the product of the RseP-dependent proteolysis has not been identified. In vivo, the N-terminal cleavage product appears to be degraded further by some cytosolic proteases, such as ClpXP (Flynn et al, 2003). Before gaining any deeper insights into the 'RIP' reaction of RseP, it is essential to establish biochemically that RseP is indeed a protease, to characterize the cleavage reaction products at the amino-acid residue level, and to reveal the substrate specificity and amino-acid sequence requirement of the proteolysis. In this work, we addressed these questions by undertaking in vivo and in vitro experimental approaches. Our results establish that RseP has a proteolytic activity against a broad range of TM sequences, including that of RseA. Results HA-MBP-RseA140 allows detection of the cytoplasmic product after its RseP-dependent site-2 cleavage Extracytoplasmic stresses result in sequential cleavages of RseA. First DegS cleaves a periplasmic region (site-1 cleavage). The product of the first cleavage (RseA(ΔP)) is then degraded RseP-dependently (Alba et al, 2002; Kanehara et al, 2002). RseP is believed to introduce the second cleavage (site-2 cleavage) into RseA at a TM or cytosolic region. RseA140 (Figure 1A), an RseA-derivative lacking most of the periplasmic domain, bypasses the site-1 cleavage requirement of the site-2 cleavage (Kanehara et al, 2002) (see Figure 2D). Although RseP is likely to introduce a single cut into RseA140 (or into RseA(ΔP)), the expected N-terminal product of its cleavage has not been detected in vivo, probably due to its rapid degradation by cytoplasmic proteases (Flynn et al, 2003). Figure 1.Proteolytic substrates of RseP used in this study. (A) Schematic representations of RseA derivatives and other RseP substrates. Regions having RseA-derived sequence are shown in white, MBP in black, LacY-derived sequence in dark gray and Bla-derived sequence striped. (B) The amino-acid sequences of the TM region of RseA (RseATM), the first (LacYTM1) and the fifth (LacYTM5) TM region of LacY and the signal peptide of Bla (SSBla) used in the above constructs. Download figure Download PowerPoint Figure 2.RseP-dependent cleavage of RseATM, LacYTM1 and LacYTM5 having N-terminal HA and MBP. (A) Immunological detection of the cleaved N-terminal products. Plasmids pSTD797 (encoding HA-MBP-RseA140; RseATM), pSTD835 (HA-MBP-RseA(LacYTM1)140; LacYTM1) and pSTD843 (HA-MBP-RseA(LacYTM5)140; LacYTM5) were introduced into a ΔrseA strain (AD1811, lanes 1, 5 and 9), as well as into its derivatives additionally deleted for rseP (KK211, lanes 2, 6 and 10), degS (AD1839, lanes 3, 7 and 11) or both (AD1840, lanes 4, 8 and 12). Plasmid-bearing cells were grown in L broth containing 1 mM IPTG and 1 mM cAMP at 30°C. Proteins were analyzed by SDS–PAGE and anti-HA (αHA) or anti-MBP (αMBP) immunoblotting. The open and closed arrowheads indicate RseP-uncleaved (UC) and -cleaved (CL) forms of each protein. (B) Membrane integration of HA-MBP-RseA140 and its derivatives. Plasmids pSTD797 (RseATM), pSTD800 (LacYTM1) and pSTD843 (LacYTM5) were introduced into KK374 (ΔrseA ΔrseP ΔdegS). Cells were grown as in (A) and converted to spheroplasts, which were treated with 1 mg/ml proteinase K (PK) in the presence or absence of 1% Triton X-100 (TX) as indicated. Proteins were analyzed by SDS–PAGE and anti-HA (αHA), anti-MBP (αMBP) or anti-FtsH (αFtsH) immunoblotting. A cytoplasmic membrane protein FtsH with a large cytoplasmic domain and a protease-resistant periplasmic region was used as an internal control for the spheroplast integrity. The open arrow indicates the intact form of each protein. (C) Pulse-chase assessment of stability of HA-MBP-RseA140 and its derivatives. Cells of AD1811 (rseP+) and KK211 (ΔrseP), each carrying pSTD797 (RseATM), pSTD835 (LacYTM1) or pSTD843 (lacYTM5), were grown in M9-glucose (0.4%) medium supplemented with 18 amino acids (other than methionine and cysteine) at 30°C and induced with 1 mM IPTG and 1 mM cAMP for 10 min for the expression of cloned genes. Cells were pulse-labeled with [35S]methionine for 2 min and chased with unlabeled methionine for the indicated periods. Proteins were then precipitated with anti-HA antibodies, separated by SDS–PAGE and visualized by a phosphor imager (BAS1800). (D) RseP-dependent degradation of HA-RseA140 and its derivatives without the MBP moiety. Plasmids pKK58 (encoding HA-RseA140; RseATM), pSTD760 (encoding HA-RseA(LacYTM1)140; LacYTM1) and pSTD767 (encoding HA-RseA(LacYTM5)140; LacYTM5) were introduced into the strains described in (A). Proteins were analyzed as in (A) by anti-HA immunoblotting. Download figure Download PowerPoint We attempted to detect a site-2-cleaved product, by replacing the cytoplasmic RseA moiety of HA-RseA140 by maltose binding protein (MBP), a stably folding domain. The resulting chimeric protein, HA-MBP-RseA140 (Figure 1A), was induced in a ΔrseA strain additionally carrying the ΔrseP and/or ΔdegS mutations (note that degS and rseP are dispensable in the presence of an rseA disruption; Alba et al, 2002; Kanehara et al, 2002) (Figure 2). HA-MBP-RseA140 was detected as the full-length product in the ΔrseP strains by anti-HA or anti-MBP immunostaining (Figure 2A, lanes 2 and 4). It was digested by proteinase K externally added to spheroplasts, generating a fragment with HA and the MBP antigenecity (Figure 2B, lanes 1 and 2). After solubilization of the membrane with a detergent, the HA part disappeared while a protease-resistant MBP domain remained (Figure 2B, lanes 3). These results indicate that HA-MBP-RseA140 is membrane integrated with an Nin–Cout orientation. In the rseP+ strains, HA-MBP-RseA140 was detected as a smaller fragment (Figure 2A, lanes 1 and 3), which retained both the MBP and the N-terminal HA antigenecity. DegS did not affect accumulation of this fragment (Figure 2A, lanes 1–4). The HA- and MBP-containing N-terminal fragment of HA-MBP-RseA140 was also generated when the ΔrseP strain was complemented with an RseP+ plasmid but not with its protease active-site motif mutant (data not shown). Stability of HA-MBP-RseA140 was then studied by pulse-chase experiments (Figure 2C). It was stable in ΔrseP cells but was degraded rapidly in rseP+ cells. In fact, the smaller fragment was a major immunoprecipitated product in the presence of RseP. These results collectively indicate that an RseP-dependent cleavage of HA-MBP-RseA140 occurs around its membrane domain to generate the N-terminal and soluble product. We also detected an RseP cleavage product for a derivative of RseA140 having a cytoplasmic Bla domain (data not shown). The cytoplasmic domain of RseA is not essential for its proteolysis by RseP. Purification of RseP and demonstration of its proteolytic activity To characterize RseP as an enzyme, its wild-type and zinc metalloprotease motif variant (H22F) forms were derivatized by attachment of a hexahistidine and a Myc epitope to the C-terminus (RseP-His6-Myc). They were overproduced, solubilized with n-dodecyl-β-D-maltoside and purified by Ni-NTA affinity column chromatography (Figure 3A, lanes 1 and 2). Wild-type RseP, but not the H22F variant, showed low but significant activity to degrade fluorescent dye-conjugated casein (BODIPY FL casein) (data not shown). Although we attempted to purify His6-tagged RseA140, they proved intractable because of high tendency of aggregation. In contrast, we were able to purify sufficient amount of His6-MBP-RseA140 (Figure 3A, lane 3). When His6-MBP-RseA140 was incubated with purified RseP, a small fragment of identical SDS–PAGE mobility as the in vivo N-terminal product was produced (Figure 3B, lanes 1–5). This indeed was an N-terminal fragment of His6-MBP-RseA140, since it reacted with anti-MBP and anti-His6 (data not shown) upon immunoblotting. No such fragment was observed upon incubation of His6-MBP-RseA140 alone (data not shown). Production of this N-terminal fragment was inhibited when the reaction mixture was supplemented with a metal chelator, 1,10-phenanthroline (Figure 3B, lanes 6 and 7). Also, the H22F mutant form of RseP was inactive in the proteolytic conversion of His6-MBP-RseA140 (Figure 3B, lanes 8 and 9). These results demonstrate that RseP indeed has a protease activity that directly cleaves RseA without involving any other protein factors. Figure 3.In vitro proteolytic reactions catalyzed by RseP. (A) Purified preparations of RseP-His6-Myc and the model substrate proteins. A 0.35 μg portion of each of purified samples of RseP-His6-Myc (lane 1), RseP(H22F)-His6-Myc (lane 2), His6-MBP-RseA140 (lane 3), His6-MBP-RseA(LacYTM1)140 (lane 4), His6-MBP-RseA(LacYTM1/P28L)140 (lane 5) and His6-MBP-RseA(A108C)140 (lane 6) was subjected to 10% SDS–PAGE and Coomassie brilliant blue (CBB) staining. Positions of molecular size markers (shown in kDa) are shown on the left. (B) Cleavage of His6-MBP-RseA140 by RseP. His6-MBP-RseA140 was incubated with RseP-His6-Myc (lanes 1–7) or RseP(H22F)-His6-Myc (lanes 8 and 9) in the presence (lanes 6 and 7) or absence (lanes 1–5, 8 and 9) of 5 mM 1,10-phenanthroline (PT) for the indicated times. 10% SDS–PAGE patterns are shown by CBB staining (upper panel) and anti-MBP immunoblotting (lower panel). (C) Cleavage of His6-MBP-RseA(LacYTM1)140 and its P28L derivative. RseP-His6-Myc was incubated with His6-MBP-RseA(LacYTM1)140 (lanes 1–5) and His6-MBP-RseA(LacYTM1/P28L)140 (lanes 6 and 7) for the indicated times followed by 10% SDS–PAGE and CBB staining. (D) MalPEG modification of RseP cleavage products of His6-MBP-RseA140 and His6-MBP-RseA(A108C)140. RseP-His6-Myc was incubated with His6-MBP-RseA140 (lanes 1–4) and His6-MBP-RseA(A108C)140 (lanes 5–8) for the indicated times. Proteins were then precipitated by trichloroacetic acid treatment, solubilized in 1% SDS and subjected to modification with 5 mM malPEG at 37°C for 1 h. They were analyzed by SDS–PAGE and anti-MBP immunoblotting. Download figure Download PowerPoint Determination of the site of RseP cleavage in RseA We then attempted to determine the RseP cleavage site in RseA. We introduced a cysteine residue into various positions of HA-MBP-RseA140 and asked whether the cysteine was retained in the cleaved product. The existence of cysteine was determined by its modification with methoxypolyethylene glycol 5000 maleimide (malPEG) (Figure 4), which adds a molecular mass of ∼5 kDa to the polypeptide, and retards its SDS–PAGE mobility. Figure 4.Determination of the RseP cleavage sites within RseATM and LacYTM1. (A) MalPEG modification of engineered cysteines before and after the RseP-dependent cleavage of RseATM variants. Cells expressing an HA-MBP-RseA140 derivative having an engineered cysteine residue in its TM sequence or in the MBP domain were grown in L broth containing 1 mM IPTG and 1 mM cAMP at 30°C. Total cellular proteins were solubilized in 1% SDS, and subjected to modification with 5 mM malPEG at room temperature for 30 min. Subsequently, samples were mixed with an equal volume of 2 × SDS sample buffer and analyzed by SDS–PAGE and anti-HA immunoblotting. The numbering of the amino-acid residues in the RseATM sequence region is according to that of the original RseA protein. (B) MalPEG modification assay to determine the cleavage site of LacYTM1. Cells expressing an HA-MBP-RseA(LacYTM1)140 derivative having an engineered cysteine residue were grown, treated with malPEG and analyzed by SDS–PAGE and anti-HA immunoblotting as in (A). The numbering of the amino-acid residues in the LacYTM1 sequence region is according to that of the original LacY protein. UC and CL indicate the uncleaved and cleaved forms, respectively, of HA-MBP-RseA and its derivatives. The open vertical arrows indicate the site of the RseP cleavage determined from the present results. The LacYTM1 and LacYTM5 sequences with amino-acid residues in a flanking MBP/linker-derived region (shown in italic) are shown on the top of each panel. Download figure Download PowerPoint HA-MBP-RseA140 contains a single cysteine (Cys109) within the segment assigned as TM (Figures 1B and 4A). HA-MBP-RseA140 and its variant, HA-MBP(Cys)-RseA140 having an additional cysteine in the MBP domain, were expressed in the rseP+ and the ΔrseP strains. Total cellular proteins were then acid-precipitated and solubilized with SDS in the presence or absence of malPEG (Figure 4A). The full-length forms of HA-MBP-RseA140 and HA-MBP(Cys)-RseA140 (in the ΔrseP cells) exhibited different extents of malPEG-induced gel mobility shifts, approximately by 5 and 10 kDa, respectively. This was as expected because these proteins contained one and two cysteine residues. The RseP-cleaved product of HA-MBP(Cys)-RseA140 (in the rseP+ cells) exhibited a mobility shift of about 5 kDa when treated with malPEG. This should have been due to modification of the introduced cysteine residue in the MBP domain, because the cleavage product of HA-MBP-RseA140 (no cysteine in MBP) was not gel-shifted. In other words, the product of the RseP cleavage did not contain Cys109. The cleavage by RseP should occur on the N-terminal side of Cys109. We then constructed a series of HA-MBP-RseA140 derivatives having a cysteine substitution in the RseA segment of Trp97 to Ala108 in its predicted TM domain (Figure 4A). All the mutant proteins thus constructed were susceptible to RseP cleavage, although the cleavage efficiencies varied somewhat. Importantly, all the N-terminal cleavage products including that of the HA-MBP-RseA140 derivative carrying an A108C substitution were modifiable with malPEG, indicating that they retained the RseA residues 97–108. We also examined malPEG modifiability of in vitro RseP reaction products of purified wild-type His6-MBP-RseA140 and its A108C mutant form. Unlike the in vitro-generated N-terminal fragment of wild-type HA-MBP-RseA140, that of the A108C mutant protein was modified with malPEG (Figure 3D, lanes 4 and 8). While the lack of Cys109 in the in vivo-generated fragment could still be explained in terms of some other proteases that secondly remove it, the in vitro results with purified materials more directly indicate that RseP catalyzes the cleavage of the peptide bond between positions 108 and 109. Taken together, our results suggest that the RseP-catalyzed cleavage occurs between Ala108 and Cys109, well inside the predicted TM sequence of RseA. Ability of RseP to cleave diverse TM sequences Thus far RseA is the only known substrate of RseP in E. coli. The results presented above suggest that RseP can cleave both Ala–Cys and Cys–Cys bonds. Does RseP recognize uniquely the RseA TM sequence as a substrate of proteolysis? To examine whether RseP can cleave other TM sequences, we constructed two variants of HA-MBP-RseA140, HA-MBP-RseA(LacYTM1)140 and HA-MBP-RseA(LacYTM5)140, by replacing the TM sequence of RseA with sequences derived from the first and the fifth TM segments of LacY, respectively (Figure 1). The results of protease accessibility tests indicated that these proteins are integrated into the membrane with the same orientation as HA-MBP-RseA140 (Figure 2B, lanes 4–9). Their accumulation and stability in rseP+ and ΔrseP cells were studied by immunoblotting (Figure 2A, lanes 5–12) and pulse-chase (Figure 2C, middle and lower panels) experiments. Their behaviors in the presence or absence of RseP were very similar to those of the original HA-MBP-RseA140 protein. Moreover, RseP cleaved His6-MBP-RseA(LacYTM1)140 in the purified reaction system, generating a fragment of the latter that was very similar to the in vivo product (Figure 3C). Thus, these model membrane proteins are cleaved by RseP as efficiently as the original protein despite the fact that they do not retain any TM sequence of RseA. HA-RseA140 derivatives (without the MBP domain) having either LacYTM1 or LacYTM5 were also degraded in vivo in an RseP-dependent manner (Figure 2D). Although some strains, such as MC4100, supported only poor degradation of HA-RseA(LacYTM1)140 and RseA(LacYTM5)140, overproduction of RseP markedly stimulated their degradation (data not shown). The RseP cleavage site in HA-MBP-RseA(LacYTM1)140 was determined again by Cys-scanning mutagenesis combined with malPEG modification (Figure 4B). In this case, LacYTM1 had no cysteine residue (Figure 1B) and each of the constructed mutant proteins contained a unique cysteine. The cleaved products of the F12C and F15C variants as well as HA-MBP(Cys)-RseA(LacYTM1)140 were modifiable with malPEG, but those of the F17C and F21C variants were not. The F16C substitution gave an intermediate result, giving rise to both modified and unmodified N-terminal fragments, the former being predominating. Thus, a major cleavage site of HA-MBP-RseA(LacYTM1)140 may be between Phe16 and Phe17, whereas the substrate is also cleaved between Phe15 and Phe16 to some extent. In addition, a very minor cleavage might occur after Phe18, since a trace of malPEG-modified N-terminal fragment was observed for the F18C substitution. LacYTM5 has two cysteine residues (Figure 1B) and the malPEG modification assay showed that both of these residues were included in the cleaved N-terminal fragment (data not shown) of HA-MBP-RseA(LacYTM5)140, indicating that the cleavage occurs within the C-terminal half of LacYTM5. These results show that RseP can act against TM sequences other than that of RseA. HA-MBP-RseA(LacYTM1)140 still contains an RseA-derived short sequence in its periplasmic region. We replaced this sequence with a combination of those from the first periplasmic loop of LacY and the Myc epitope (Figure 1A). The resulting protein, HA-MBP-LacYTM1-P1*-Myc, was mostly membrane-integrated (Figure 5A, lanes 5–7) and cleaved by RseP (Figure 5A, lanes 1–4). Thus, RseP can exert its proteolytic activity without involving any amino-acid sequence of RseA. Figure 5.RseP-mediated cleavage of proteins having no RseA-related sequence. (A) RseP-dependent cleavage and topology of HA-MBP-LacYTM1-P1*-Myc. Lanes 1–4: plasmid pSTD853 was introduced into strains AD1811 (ΔrseA, lane 1), KK211 (ΔrseA ΔrseP, lane 2), AD1839 (ΔrseA ΔdegS, lane 3), AD1840 (ΔrseA ΔrseP ΔdegS, lane 4) and KK374 (ΔrseA ΔrseP ΔdegS, lanes 5–7). In vivo cleavage of HA-MBP-LacYTM1-P1*-Myc by RseP (lanes 1–4) and its topology in the membrane (lanes 5–7) were analyzed by SDS–PAGE and anti-HA immunoblotting as described in the legend to Figure 2. P1* indicates a region derived from the LacY first periplasmic loop, with amino-acid substitutions introduced into two positions that fortuitously have the same amino-acid residues as the RseA periplasmic residues of the corresponding locations. (B) RseP-mediated cleavage of HA-MBP-SSBla-Bla. Total proteins prepared from cells of AD1811 (ΔrseA, lane 1), KK211 (ΔrseA ΔrseP, lane 2), AD1839 (ΔrseA ΔdegS, lane 3) and AD1840 (ΔrseA ΔrseP ΔdegS, lane 4), each carrying pSTD849, were analyzed as above. HA-MBP-SSBla indicates the leader peptidase-cleaved product of the full-length protein, whereas its RseP cleavage product is indicated as HA-MBP-SSBla*. Download figure Download PowerPoint We also examined whether RseP can cleave a signal sequence of a secretory protein, β-lactamase. To facilitate detection of such cleavage, HA-MBP domain was fused to the N-terminus of the β-lactamase precursor (Figure 1A and B). When this fusion protein was expressed in the ΔrseP cells, its full-length product (HA-MBP-SSBla-Bla) and a smaller fragment (HA-MBP-SSBla) were detected by anti-HA and anti-MBP immunostaining (Figure 5B). The small fragment that did not react with anti-Bla antibodies (data not shown) most probably represented a product of leader peptidase (Lep) cleavage of the fusion protein on the periplasmic side since a secA51(Ts) mutation causing a defect in protein translocation prevented its generation (data not shown). The same protein in the rseP+ cells also produced two products, among which the smaller product (HA-MBP-SSBla*) was further downshifted as compared to the Lep cleavage product seen in the ΔrseP strain (Figure 5B, lanes 1 and 2). Thus, the HA-MBP signal peptide was cleaved in an RseP-dependent manner. MalPEG treatment caused about a 5 kDa upshift of HA-MBP-SSBla, whereas it did not affect the mobility of HA-MBP-SSBla* (data not shown), suggesting that the cleavage occurred N-terminally to the unique Cys residue in the Bla signal sequence (Figure 1A). We suggest that RseP can introduce a proteolytic cleavage into the signal sequence of β-lactamase. Sequence features that accept a cleavage by RseP We then addressed what features of a TM sequence make it susceptible to proteolysis by RseP (Figure 6). We chose LacYTM1 as a target of systematic mutagenesis because its structure is known at least as a part of the native protein (Abramson et al, 2003). In the following mutation studies, we first tested whether constructed mutant proteins assembled into the membrane with the unaltered orientation, and only those that indeed did so (see Figure 6B, lanes 4 and 5) were subjected to further characterization. We first mutated the unique proline residue (Pro28) in the C-terminal region of the LacYTM1 segment in HA-MBP-RseA(LacYTM1)140. A leucine substitution for Pro28 severely impaired the cleavage by RseP (Figure 6B, lanes 1 and 2 of M1). Glycine and serine at this position also compromised the cleavage reaction (Figure 6B, M2 and M3) but not as severely as leucine. Asparagine here moderately affected the cleavage (Figure 6, M4). It was reported that leucine, phenylalanine, glycine, tyrosine, serine, asparagines and proline have helix-forming propensity decreasing in this order in a membrane-mimicking environment, with proline being the lowest among all the amino acids (Liu and Deber, 1998). Thus, the inhibitory effects of the substitutions at position 28 seem to be correlated with their helix-forming abilities. We also examined possible contribution of glycine (Gly13 and Gly25) and alanine (Ala26) residues in the middle of LacYTM1. A G13L substitution and a combination of G25L and A26L substitutions each exerted a weak effect on cleavage (data not shown). However, when all of these three mutations were combined (Figure 6B, M6), a strong cleavage defect was observed. The mutant protein M6 contains five leucines altogether in its TM region. An additional F17P substitution introduced into this protein partially restored the cleavage in rseP+ cells (Figure 6, M7). Similar proline effects were observed at different positions (positions 19 and 20; Figure 6B, M8 and M9). Glycine substitution for Tyr19 was virtually ineffective (Figure 6B, M10), while either serine (Figure 6B, M11) or asparagine (Figure 6B, M12) at this position allowed significant restoration of cleavage. Glycine and tyrosine have similar helix-forming propensity, higher than that of serine or asparagine (Liu and Deber, 1998). These results suggest that helix-destabilizing residues promote cleavage of TM segments by RseP. It should be noted that leucine at position 28 appeared to be particularly destructive, since the Y19P substitution caused little improvement in cleavage efficiency for the M1 mutant (Figure 6B, M5). Figure 6.Effects of LacYTM1 amino-acid substitutions on the cleavage by RseP. (A) Amino-acid sequences of the TM domains of the HA-MBP-RseA(LacYTM1) derivatives. % cleavage on the right represents the proportion of the RseP-cleaved form in the sum of