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

Akira Saitoa, Yohei Hizukuria, Ei-ichi Matsuob, Shinobu Chibaa,1, Hiroyuki Moria, Osamu Nishimurab, Koreaki Itoc, and Yoshinori Akiyamaa,2

aInstitute for Virus Research, Kyoto University, Kyoto 606-8507, Japan; bDivision of Disease Proteomics, Institute for Research, Osaka University, Osaka 565-0871, Japan; and cFaculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan

Edited by Linda L. Randall, University of Missouri, Columbia, MO, and approved July 11, 2011 (received for review May 25, 2011) A signal (SP) is cleaved off from presecretory by toplasmic membrane, in contrast with the currently prevailing during or immediately after insertion into the concept that SppA (12, 13), a protease unrelated to the S2P membrane. In metazoan cells, the cleaved SP then receives pro- or the SPP family, is the responsible for teolysis by signal peptide peptidase, an intramembrane-cleaving cleavage in bacteria. protease (I-CLiP). However, bacteria lack any signal peptide pepti- dase member I-CLiP, and little is known about the metabolic fate of Results bacterial SPs. Here we show that Escherichia coli RseP, an site-2 RseP-Dependent in Vivo Cleavage of β-Lactamase SP. The secretory protease (S2P) family I-CLiP, introduces a cleavage into SPs after precursor of Bla has a signal peptide of 23 residues. We char- their signal peptidase-mediated liberation from preproteins. A Ba- acterized observed with HA-MBP-SPBla-Bla (11), cillus subtilis S2P protease, RasP, is also shown to be involved in SP having a hemagglutinin (HA)–maltose-binding protein (MBP) cleavage. These results uncover a physiological role of bacterial S2P domain (HA–MBP domain) attached to the N terminus of the and update the basic knowledge about the fate of signal Bla precursor (Fig. 1A). Immunoblotting using anti-HA detected peptides in bacterial cells. two HA-MBP-SPBla-Bla bands in ΔrseP cells (Fig. 1B, lane 2): the full-length protein (Full) and a smaller species designated intramembrane proteolysis | signal sequence uncleaved (UC). Notably, rseP+ cells produced an additional and prominent, faster-migrating band designated cleaved (CL) (lane MICROBIOLOGY signal peptide (SP) at the N terminus of secretory protein 1 in Fig. 1B). Pulse-labeling and anti-HA immunoprecipitation fi A precursors (preproteins) is cleaved off by signal (leader) experiments veri ed the above observations (Fig. S1, Lower, peptidase (1) and left behind in the membrane, typically as- lanes 2 and 4). Production of UC and CL was diminished markedly (Fig. S1, Lower, lanes 1 and 3) by treatment of cells suming the type II (Nin-Cout) transmembrane configuration. In mammalian cells, the liberated SPs receive further cleavage by with NaN3, a SecA inhibitor, which prevented translocation/ signal peptide peptidase (SPP) and are released from the mem- maturation of envelope proteins (Fig. S1, Upper, OmpA), as well brane (2–4). Small peptides derived from SP sometimes act as as by the lep-9 mutation affecting Lep, the major E. coli signal a regulatory molecule (3). (leader) peptidase (Fig. 1D; see below). SPP belongs to intramembrane-cleaving proteases (I-CLiPs), The in vivo production of CL (presumably from UC) required which are classified into SPP/γ-secretase (aspartyl proteases), the protease activity of RseP, as it took place in cells expressing rhomboid (serine protease), and S2P (zinc metalloprotease) (5, 6). the wild-type enzyme (RseP) but not in cells expressing an active- These proteases liberate otherwise membrane-tethered domains of site mutant form of RseP [RseP(H22F)] with an alteration in the H22ExxH motif (Fig. 1C). These results suggest that UC repre- membrane proteins to function as a regulatory molecule. I-CLiPs Bla have specific intramolecular routes that make water molecules sents the N-terminal HA-MBP-BlaSP segment (HA-MBP-SP ) generated by Lep and that CL is produced from UC by RseP- accessible to the intramembrane proteolytic active sites (5, 6). For Bla γ mediated proteolysis within SP . In the above experiments, such proteolysis to occur, -secretase/SPP and S2P require, in most fi Bla cases, removal of the ectodomain of the substrate by other protease a signi cant portion of HA-MBP-SP -Bla accumulated as the full-length form. Probably, this resulted from inefficient targeting (5, 7). SPP and S2P prefer substrate with the type II trans- – membrane orientation and γ-secretase and rhomboid prefer the of the model protein having the N-terminally attached HA MBP type I orientation (7). domain, but not from jamming of the translocon with overex- Bacteria contain S2Ps and rhomboids but not SPPs, and only pressed fusion protein, as proOmpA processing was not appre- limited knowledge is available about the fate of bacterial SPs (7). ciably affected (Fig. 1D and Fig. S1). We and others have been characterizing RseP, an Escherichia . σE RseP Directly Cleaves Synthetic Bla SP Peptides in Vitro We designed coli member of the S2P involved in the pathway extrac- a chemically synthesized substrate, Myc-SPBla-Flag, in which the ytoplasmic stress response (8, 9). In this regulation, a protease, Bla SP (SPBla) was sandwiched between Myc and Flag sequences DegS, responds to misassembled outer membrane proteins and fi (Fig. 2A). The extra sequences were anticipated to increase introduces the rst proteolytic cleavage into RseA, a membrane- solubility and facilitate detection of the peptide. Incubation of integrated anti-σE protein (10). Subsequently, RseP introduces the second cleavage into RseA, activating σE to transcribe stress- inducible genes (8, 9). Although RseA is the only physiological Author contributions: Y.A. designed research; A.S., Y.H., E.-i.M., S.C., H.M., O.N., and Y.A. substrate of RseP so far established, we have shown previously performed research; A.S., Y.H., E.-i.M., S.C., K.I., and Y.A. analyzed data; and K.I. and Y.A. that RseP can cleave a wider range of transmembrane sequences wrote the paper. having helix-destabilizing residues (11). Our preliminary results The authors declare no conflict of interest. β that RseP cleaved a -lactamase (Bla) fusion protein at or This article is a PNAS Direct Submission. around its SP (11), together with the fact that RseP and SPP 1Present address: Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, share the substrate preference for the type II orientation, Japan. prompted us to undertake the present study. Our results suggest 2To whom correspondence should be addressed. E-mail: [email protected]. strongly that S2Ps (E. coli RseP and Bacillus subtilis RasP) are This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. involved in degradation of remnant SPs left in the bacterial cy- 1073/pnas.1108376108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1108376108 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 RseP Cleaves Bla SP Within the Hydrophobic Core. We extracted the A Lep B (kDa) Periplasm 12 F1 and F2 peptides from the gel bands and determined their Full 81.8 Bla RseP identities by mass spectrometric (MS) analysis (Fig. S3). The MS 68.4 SP Cytoplasm 55.0 spectra of F1 and F2 displayed a major peak of m/z = 2,596.49 MBP UC A B m/z C CL (Fig. S3 and ) and = 2,461.13 (Fig. S3 ), which repre- UC CL 41.6

HA rseP + sented the N-terminal 22 residue fragment (N22) and the C-terminal 21 residue fragment (C21) of Myc-SPBla-Flag, re- fi C D 123 456 spectively. MS/MS analysis con rmed their sequence identities 123(kDa) p OmpA (Fig. S3 D and E). Also, MS analysis of F3 and F4 (Fig. S2 C and RseP m 41.6 D) showed that these bands corresponded to the N-terminal 12- 81.8 Full Full residue fragment and the C-terminal 21-residue fragment of UC Bla UC SP -Flag, respectively. These results indicate that RseP prin- CL 41.6 CL pRseP Chase (min) 0 2 4 024 cipally hydrolyzes the Pro12-Phe13 peptide bond of Bla SP WT vec + in vitro.

H22F lep lep-9 We then characterized RseP-dependent cleavage of Bla SP Fig. 1. RseP-dependent cleavage of HA-MBP-SPBla-Bla. (A) Schematic of HA- fi Bla in vivo by cysteine scanning-modi cation experiments (Fig. 3) MBP-SP -Bla and its cleavage by Lep and RseP. (B) Detection of cleavage (11). A series of derivatives having an engineered and unique products. Cells of AD1811 (rseP+) (lane 1) and KK211 (ΔrseP) (lane 2), each carrying pSTD849 (HA-MBP-SPBla-Bla), were grown at 30 °C in L broth that cysteine at different positions within the SP were constructed and contained IPTG and cAMP for induction of HA-MBP-SPBla-Bla. Total cellular CL was examined to see whether it contained the introduced proteins were analyzed by SDS/PAGE and anti-HA immunoblotting. Intact cysteine. We used modifiability with methoxypolyethylene glycol HA-MBP-SPBla-Bla (Full) and putative RseP-uncleaved (UC) and RseP-cleaved 5000 maleimide (malPEG), a thiol-alkylating reagent of about (CL) forms of Lep-processed HA-MBP-SPBla are indicated. (C) The UC-to-CL 5 kDa, to assess the presence of cysteine. HA-MBP-SPBla-Bla conversion depends on proteolytic activity of RseP. Strain AD2328 (ΔrseP)/ derivatives with a cysteine at position 9, 12, or 15 produced UC pSTD849 was transformed further with pKK12 (RseP, lane 1), pTH18cr [vector in ΔrseP cells and both CL and UC in rseP+ cells. Whereas CLs (vec), lane 2], or pAS90 [RseP(H22F), lane 3] and analyzed by anti-RseP and – – anti-HA immunoblotting. (D) Lep dependence of the RseP-dependent from the Cys-12 (Fig. 3, lanes 5 8) and Cys-15 (Fig. 3, lanes 9 cleavage of Bla SP. Cells of IT42 (lep+, lanes 1–3) and IT41(lep-9, lanes 4–6), 12) variants were unmodifiable, that from the Cys-9 variant (Fig. each carrying pSTD849, were grown in M9 medium first at 32 °C and then at 3, lanes 1–4) received malPEG modification (see Fig. S4 for 42 °C for 20 min, induced with isopropyl 1-thio-β-D-galactopyranoside (IIPTG) controls and additional data). Thus, the in vivo-produced CL and cAMP for 10 min, and pulse-labeled with [35S]methionine for 1 min should have contained the N-terminal 9 residues of Bla SP but followed by chase. Labeled proteins were precipitated with anti-OmpA not the 12th residue and its C-terminal side. Thus, an RseP (Upper panels) and anti-HA (Lower panels). cleavage point in vivo may lie somewhere between the 9th and the 12th residues of Bla SP. Although this assignment deviates slightly from the cleavage site determined in vitro (see Discussion the synthetic peptide with purified RseP-His6-Myc (11) in de- tergent at 37 °C resulted in the generation of smaller fragments for possible cause of this apparent discrepancy), our in vivo and (designated F1 and F2) at the expense of the full-length substrate in vitro results collectively indicate that RseP cleaves Bla SP (Fig. 2B, lanes 1–4). This conversion depended on the proteolytic within the central hydrophobic region. activity of RseP because it was not observed with the purified RseP RseP Cleavage of SP Requires a Preceding Processing of Preproteins (H22F)-His -Myc (lanes 7 and 8) and was inhibited with 1,10- 6 by Lep. We addressed whether Lep-mediated SP processing is phenanthroline, a Zn2+ chelator (lanes 9 and 10). Thus, RseP a prerequisite for the cleavage of SP, using the lep-9(Ts) muta- directly cleaved Myc-SPBla-Flag. We also examined whether RseP tion that compromises the Lep activity (14) (Fig. 1D). Pulse- acted against SPBla-Flag having no N-terminal tag (Fig. S2A). This chase experiments showed that the Lep-mediated processing of peptide was also cleaved by RseP, generating two fragments (F3 fi proOmpA was delayed markedly in the lep-9 mutant cells (Up- and F4) albeit at lower ef ciency due possibly to their low solu- per). Notably, the production of not only UC but also CL from bility (Fig. S2B). HA-MBP-SPBla-Bla was diminished in this mutant (Lower).

A

B Myc-SPBla-Flag 1234 56 78 910 1112 (kDa) F2 5 F1 2 Time (h) 0243 12 120 120 01212 0 WT H22F WT - +PT

Fig. 2. Proteolysis of chemically synthesized Bla SP by purified RseP. (A) sequence of Myc-SPBla-Flag. The Myc, Flag, SPBla, N22, and C21 Fig. 3. Assessment of the RseP-dependent in vivo cleavage site of HA-MBP- segments are indicated. The amino acid numbers in SPBla start from the bla SPBla-Bla. Total cellular proteins from cells of AD1811 (rseP+) and KK211 initiator methionine. (B) In vitro reaction. Substrate peptide (19.8 μM) was (ΔrseP), expressing one of the HA-MBP-SPBla-Bla variants having a single Bla incubated at 37 °C with RseP-His6-Myc (0.64 μM; lanes 1–6, 9, and 10), RseP cysteine at the indicated position of SP , were treated with or without 5 (H22F)-His6-Myc (0.64 μM; lanes 7 and 8), or buffer alone (lanes 11 and 12). mM malPEG and analyzed by SDS/PAGE and anti-HA immunoblotting. All of 1, 10-Phenanthroline (PT; 5 mM) was included as indicated (lanes 9 and the variants were derivatives of the SP-Cys-less (C18A) form of HA-MBP-SPBla- 10). Samples were analyzed by 12% Nu PAGE (Invitrogen) and Coomassie Bla that had no cysteine residue in the SP part. “Full-malPEG(3×)” indicates Brilliant Blue G-250 staining. F1 and F2 indicate cleavage products of the the full-length protein with all three cysteine residues (one in SP and two in substrate peptide. the Bla mature part) modified with malPEG.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1108376108 Saito et al. Downloaded by guest on September 30, 2021 These results suggest that proteolysis of SP by RseP requires it contribute to the RseP-independent cleavage observed with the preceding processing by Lep. M13 coat and TolC fusion proteins. The SPs examined above are targeted to the Sec translocation Generality of RseP-Dependent SP Cleavage. To address the gener- pathway. We asked whether RseP could cleave Tat pathway SPs ality of RseP-catalyzed SP cleavage, we attached HA-MBP to the carrying the twin-arginine motif in the N-terminal region (17). precursors of selected outer membrane, periplasmic and cyto- An HA-MBP-fusion of the TorA precursor, a Tat substrate, was plasmic membrane proteins of E. coli, each having a cleavable SP found to produce a UC fragment having the HA epitope in ΔrseP Δ + (Fig. S5A). In rseP cells, all of the fusion proteins produced cells and a smaller CL fragment in rseP cells (Fig. 4A, lanes 21 fragments (designated UC) that reacted with anti-HA (Fig. 4) Bla and 22). Thus, RseP appears to cleave a Tat SP as well. and had sizes similar to that of UC from HA-MBP-SP . They To examine whether the presence of the MBP domain in the were found to produce smaller fragments (designated CL) in immediate vicinity of SP played any important role in the RseP- rseP+ cells. This was shown for OmpF, LivK, SecM, PhoA, LivJ, + dependent SP cleavage that we observed in vivo, we constructed OmpC, and Lpp derivatives using rseP cells grown at 30 °C or a derivative of the OmpC precursor (preOmpC) having an HA- – 37 °C (Fig. 4A, lanes 1 14). Detection of CL from the OmpA Bla domain, instead of an HA–MBP domain, at the N terminus. derivative required overproduction of RseP, whereas RseP This fusion protein, just like HA-MBP-preOmpC, produced CL (H22F) was ineffective (Fig. 4B). Thus, RseP has the ability to in an RseP-dependent manner (Fig. 4C). Thus, MBP played no cleave SPs from these proteins in vivo. The RseP dependence on positive role in the in vivo cleavage of SPs. CL production was less strict in the cases of the M13 coat and fi TolC and RbsB constructs; in these cases, low but signi cant Involvement of B. subtilis RasP in SP Cleavage. We attached HA- Δ amounts of CL-like fragments were produced even in the rseP MBP to the N termini of the secretory precursors of PenP and cells (Fig. 4A, lanes 15–20). SPs of these proteins may be subject fi Mpr (Fig. S5)ofB. subtilis. This Gram-positive bacterium con- to low-ef ciency cleavage by some protease other than RseP. tains RasP, an S2P protease involved in transmembrane stress GlpG, the E. coli homolog of the rhomboid family I-CLiP, was signal transduction (18). In ΔrasP cells, the PenP fusion protein not involved in the RseP-independent cleavage of SPs as the produced a fragment that reacted with anti-MBP (Fig. 5A, lane ΔglpG mutation exerted no apparent effect on the production of + 2), most likely representing UC. By contrast, rasP cells accu- the CL-like fragment (Fig. S5B). mulated a smaller sized fragment (CL, lane 1). UC in ΔrasP cells Earlier studies showed that a membrane-bound protease was converted to CL upon expression of proteolytically active SppA degraded SP from Lpp in detergent extracts (15, 16). MICROBIOLOGY RasP (RasP-His ) (Fig. 5B, lane 1) but not of its active-site However, its in vivo function has not been established. We 6 expressed HA-MBP–tagged derivatives of Lpp, LivK, M13 coat, mutant, RasP(E21A)-His6 (lane 2). The Mpr fusion protein be- and TolC in strains deleted for one or both of rseP and sppA (Fig. haved similarly (Fig. 5 A and B, lanes 3 and 4). Thus, RasP can S5C). Disruption of sppA did not significantly affect the pro- cleave SPs from at least certain secretory proteins in B. subtilis. duction of CL and CL-like fragments from any of the above con- RseP Can Cleave an Authentic Signal Peptide in the Membrane. We structs. Thus, SppA had no positive role in the RseP-dependent then studied proteolysis of a native, unmodified SP using in vitro cleavage of SPs from the Lpp and the LivK fusion proteins, nor did translocation system. A model secretory protein, LivK-Lpp, having the LivK SP region followed by the Lpp mature sequence (Fig. 6A), was synthesized by in vitro in the presence 12 3 4 5 6 78 35 A UC of [ S]methionine. It was then denatured with urea and sub- CL jected to translocation reaction into inverted membrane vesicles rseP + + + + (IMVs) prepared from wild-type (rseP+) cells. This reaction OmpF LivK SecM PhoA yielded two lower-molecular mass species of LivK-Lpp (Fig. 6B, 91011121314 UC lane 6), one corresponding to the mature (SP-processed) form CL (M) and the other (about 3 kDa) comigrating with the in vitro- rseP + + + LivJ OmpC Lpp translated LivK SP (Fig. 6B, lanes 3 and 6). The production of

UC 15 16 17 18 19 20 UC 21 22 these smaller fragments required active translocation of LivK- Lpp because they were not observed in the presence of NaN CL CL 3 rseP + + + + (lane 7) or in the absence of IMV (lane 5). Indeed, both frag- M13 coat TolC RbsB TorA ments resisted proteinase K (lane 9) unless the membrane bar-

123 B 12RseP C UC UC 1 2 UC CL CL 12 34 A B UC UC rseP + pRseP rseP + OmpA WT vec 12 34 CL CL H22F OmpC UC UC OmpA CL CL RasP rasP + + -His6 Fig. 4. Cleavage of SPs from various presecretory proteins in vivo. (A) PenP Mpr pRasP WT WT E21A – E21A Cleavage of SPs from preproteins having the N-terminally attached HA MBP PenP Mpr domain. Cells of AD1811 (rseP+; odd-numbered lanes) and KK211 (ΔrseP; even-numbered lanes), expressing HA-MBP fusions of the indicated pre- Fig. 5. Involvement of RasP in SP cleavage in B. subtilis.(A) B. subtilis strains

protein, were grown at 30 °C (lanes 1–8, 15, and 16) or 37 °C (lanes 9–14 and SCB1450 (thrC::PxylAha-mbp-penP, lane 1), SCB1628 (thrC::PxylAha-mbp-penP, 17–22) in L broth containing IPTG and cAMP for anti-HA immunoblotting. (B) ΔrasP, lanes 2), SCB1652 (thrC::PxylAha-mbp-mpr, lane 3), and SCB1673 + Cleavage of OmpA SP. (Left) The rseP (lane 1) and ΔrseP (lane 2) cells, (thrC::PxylAha-mbp-mpr, ΔrasP, lane 4) were grown at 37 °C in L broth con- carrying pAS80 encoding HA-MBP-proOmpA, were grown at 37 °C. (Right) taining 0.5% xylose to A600 of ∼0.5. Total cellular proteins were analyzed by Strain AD2328 (ΔrseP)/pAS80 was further transformed with pKK12 (RseP, SDS/PAGE and anti-MBP immunoblotting. (B) Cells of SCB2287 (thrC::PxylAha- lane 1), pTH18cr [vector (vec), lane 2], or pAS90 [RseP(H22F), lane 3] and mbp-penP, ΔrasP, amyE::PxylArasP-his6, lane 1), SCB2288 (thrC::PxylAha-mbp- grown at 37 °C. Proteins were visualized by immunoblotting with anti-RseP penP, ΔrasP, amyE::PxylArasP(E21A)-his6, lanes 2), SCB2292 (thrC::PxylAha-mbp- (Upper) and anti-HA (Lower). (C) Cleavage of HA-Bla-tagged OmpC SP. The mpr, ΔrasP, amyE::PxylArasP-his6, lane 3), and SCB2293 (thrC::PxylAha-mbp-mpr, + rseP (lane 1) and ΔrseP (lane 2) cells, carrying a pSTD1415 (HA-Bla-pre- ΔrasP, amyE::PxylArasP(E21A)-his6, lanes 4) were grown as above for anti-MBP OmpC), were grown and analyzed as in A. (Upper panels) and anti-His tag (Lower panels) immunoblotting.

Saito et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 A authentic SP in the membrane, although we have been unable to detect cleavage products due presumably to their small sizes. Discussion B (kDa) 12345678910 We have shown that bacteria use an I-CLiP protease, S2P, to 10 P introduce a proteolytic cleavage into signal (leader) peptidase- * ** M 5 SP processed SPs. In support of this conclusion, an E. coli mutant 2 lacking RseP and a B. subtilis mutant lacking RasP accumulated SP+ 3 SP SP 0 5 5 5 5 PK PK uncleaved SPs, whereas wild-type bacteria produced shorter -

Az Az* Tx 3 fragments of SPs. This demonstration required the use of no IMV + IMV engineered preproteins having an N-terminally attached HA- (kDa) MBP or HA-Bla domain, which allowed the detection of SPs 1 2345 678910 C 10 P and their cleaved N-terminal fragments. The stable MBP-Bla M 5 domains might also have stabilized the short hydrophobic oli- SP 2 gopeptides. However, these globular domains did not have any Time (min)0 12 24 36 48 0122436 48 essential role in the RseP-dependent proteolysis of SP per se. IMV rseP+ rseP This conclusion was supported by the results of our in vitro studies showing that (i) the purified RseP enzyme directly D 123 456 789(kDa) cleaved the synthetic Bla SP with or without the short N-ter- P 10 minal Myc tag and (ii) the unmodified LivK SP received RseP- M dependent proteolysis in the membrane when it was produced in 5 SP 2 the in vitro translocation reaction. Time (min) 0124 0124 0124 RseP cleaved Bla SP in vitro between Pro-12 and Phe-13 within IMV WT +RseP +H22F the hydrophobic core. However, position 12 that had been replaced by cysteine was not included in the N-terminal cleavage Fig. 6. Cleavage of an authentic SP by RseP. (A) Amino acid sequence of product that we detected in vivo. This apparent discrepancy could the N-terminal region of LivK-Lpp. The Lep cleavage site is indicated by an be explained in different ways. First, the cleavage point may have arrowhead. (B) Detection of LivK SP generated upon in vitro translocation. 35 shifted due either to the cysteine substitution or the attachment of [ S]methionine-labeled and urea-denatured LivK-Lpp was incubated at the HA–MBP domain. Second, some secondary proteolysis may 30 °C for the indicated periods in the translocation reaction mixture with or + have occurred in vivo. Third, enzyme-substrate positioning may without IMVs prepared from AD2466 (rseP ). NaN (Az; 50 mM) or a mix- 3 have differed between the membrane-integrated states (in vivo) ture (Az*) of NaN3 (50 mM), NaCl (1 M), and zinc acetate (50 μM) was in- cluded in the reactions for lanes 7 and 8. A portion of the sample for lane 6 and the detergent-solubilized states (in vitro) of the substrate and was treated with 200 μg/mL proteinase K in the presence (lane 10) or ab- the enzyme. Although further studies are required to establish the sence (lane 9) of 1% Triton X-100. Lanes 1–3 received in vitro-synthesized exact peptide bond that RseP hydrolyzes in vivo, our results col- peptides (open arrowheads) of the first 20 (lane 1, SP-3), 26 (lane 2, SP+3), lectively indicate that RseP cleaves the Bla SP within the hydro- and 23 (lane 3, SP) residues of the LivK-Lpp sequence, which were in- phobic core region. In addition to the Bla SP, SPs from 12 E. coli cubated with IMVs. Proteins were analyzed by 10–20% tricine gel electro- preproteins proved to be subject to RseP-dependent cleavage. The phoresis. Arrow indicates a fragment of the SP+3 peptide possibly RseP-susceptible SPs include those of both Sec and Tat substrates, produced by degradation or premature translation termination. Asterisks pointing to the general involvement of RseP in SP metabolism indicate possible read-through products. (C) Effects of rseP disruption. Urea-denatured LivK-Lpp was incubated as above with IMVs from AD2466 in E. coli. (rseP+,lanes1–5) or from AD2469 (ΔrseP,lanes6–10) for 3 min at 30 °C. One of B. subtilis S2P proteases, RasP, was found to be involved After addition of azide/NaCl/zinc, samples were incubated further at 30 °C in proteolysis of SPs from at least two secretory proteins. Clear- for the indicated periods. (D) Effect of RseP overproduction. Urea-dena- ance of SPs from the membrane might be important for protein tured LivK-Lpp was incubated as above with IMVs from CU141 [(WT), lanes in this , as the B. subtilis rasP disruptant exhibits – – 1 3], from CU141/pKK49 (RseP-His6-Myc) (RseP, lanes 4 6), or from CU141/ a weak defect in protein secretion (19). In accordance with our – pKK50 (RseP(H22F)-His6-Myc) (H22F, lanes 4 6) and analyzed as in C.The conclusion, it was reported that several peptide sex pheromones letters P, M, and SP indicate the precursor form, mature form, and signal in Enterococcus faecalis are produced from the lipoprotein SP peptide, respectively, of LivK-Lpp. through S2P (Eep)-dependent, endoproteolytic processing (20, 21). We suggest that S2P proteases in bacteria have a role equiv- alent to that of SPPs in higher eukaryotes. Eukaryotic S2P pro- rier was disrupted with a detergent (lane 10). Thus, the 3-kDa teases could potentially act against SPs, but their localization at fragment most likely represented the LivK SP. the Golgi apparatus (22) might preclude them from acting against We examined stability of the in vitro-produced LivK SP by SPs, which are likely to remain in the endoplasmic reticulum (ER) incubating a 6-min reaction product in the presence of NaN3 and membrane. This spatial segregation seems to explain why higher a high concentration of salt, which together blocked further eukaryotes possess SPP for degrading SPs in the ER. Both S2P translocation effectively (Fig. 6B, lane 8). The inclusion of high (a zinc metalloprotease) and SPP (an aspartyl protease) prefer salt was also known to stimulate in vitro proteolytic activity of membrane proteins with type II orientation as substrates (7), in RseP (11). During this posttranslocation incubation, the intensity which helix-destabilizing amino acid residues promote proteolysis of LivK SP in wild-type IMVs decreased gradually (Fig. 6C, lanes by these proteases (2, 11). SPs fulfill these requirements (23). fi 1–5), indicating that it was degraded or cleaved into undetectable Confusingly, the name of signal peptide peptidase was rst fragments. By contrast, LivK SP produced in the ΔrseP IMVs used for the E. coli enzyme SppA (or protease IV) (12, 15), a serine protease having no evolutionary relationship to SPP but was significantly more stable (Fig. 6C). The degradation was with the ability to degrade Lpp SP in detergent extracts and after enhanced markedly when IMVs from RseP-overproducing cells purification (12, 15, 16). Although SppA is generally considered – – were used for translocation (Fig. 6D; compare lanes 4 6with1 3) to be the SP-cleaving enzyme in bacteria, disruption of the E. coli but not with IMVs from RseP(H22F)-overproducing cells (lanes sppA gene did not affect in vivo cleavage of SPs of fusion pro- 7–9) (see Fig. S6 for quantification). These results demonstrate teins, including that from Lpp in our experiments. SppA has a successful reconstitution of RseP-dependent proteolysis of an a large periplasmic domain, which, in archaea, forms a tetra-

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1108376108 Saito et al. Downloaded by guest on September 30, 2021 meric assembly of an inverted bowl-like shape, the membrane- of SPs and RseA offers excellent systems to gain molecular and distal part of which includes the protease active sites (24). The cellular insights into the mechanisms of regulated intramembrane possibility remains that SppA provides a route of SP degradation proteolysis. if SPs are released to the periplasm, although its in vivo role has not been studied. Our observations that SPs from some proteins Materials and Methods received RseP/SppA-independent in vivo cleavage and that LivK Bacterial Strains, Plasmids, and Media. Bacterial strains are listed in Table S1. SP was slowly degraded even in the RseP-deficient membranes See SI Materials and Methods for details of strains, plasmids, and media. (Fig. 6C, lanes 6–10) suggest that RseP is not the exclusive en- zyme that degrades SPs. Identification and characterization of Immunoblotting, Pulse-Chase Labeling, and Immunoprecipitation. Immuno- additional proteases involved in SP turnover await future studies. blotting (32), pulse-chase labeling (8), and immunoprecipitation (8) analyses Our previous (11) and the present results show that the sub- were carried out essentially as described. See SI Materials and Methods strate specificity of RseP is broader than originally thought. It is for details.

possible that RseP homologs, prevalent in bacteria, contribute to Bla the quality control of the cytoplasmic membrane by degrading Cleavage of Synthetic Peptides. Chemically synthesized Myc-SP -Flag pep- tide (19.8 μM) was mixed with purified RseP-His6-Myc or RseP(H22F)-His6-Myc SPs as well as some membrane proteins. Consistent with this (0.64 μM) (11) in buffer containing 50 mM Tris·HCl (pH 8.1), 0.02% n-dodecyl- notion, RseP appears to have a function other than activation of β-D-maltoside, 2.5% glycerol, 5 μM zinc acetate, 10 mM 2-mercaptoethanol, σE fi , as disruption of rseP causes signi cantly slower cell growth and 1× protease inhibitor mixture (EDTA-free) (Nacalai Tesque, Inc.), in- even in the absence of RseA, the proteolytic target that down- cubated at 37 °C as described previously (11) and analyzed by SDS/PAGE and E regulates σ (Fig. S7). In eukaryotes, SPPs might have roles mass spectrometry. See SI Materials and Methods for details. beyond SP degradation (25, 26). However, RseP (S2P) or SPP must not promiscuously degrade membrane proteins. Cleavage In Vitro Translocation and Proteolysis of LivK SP. In vitro transcription was of SPs by SPP (2) and RseP (this study) requires prior processing directed by pSTD1411, pSTD1425, pSTD1426, and pSTD1427 and [35S]- of the substrates by the signal (leader) peptidase. The Lep de- methionine-labeled LivK-Lpp proteins were synthesized essentially as de- pendence of the RseP action should prevent premature cleavage scribed previously (33). After translation, proteins were precipitated with 5% of SPs before the commitment step of translocation. It might also trichloroacetic acid and solublized with 6 M urea in 50 mM Hepes-KOH (pH 7.5). IMVs were prepared as described previously (34). Translocation of prevent the formation of a potentially harmful protein species 35 that bears the C-terminal portions of SPs. This regulation is in vitro-synthesized S-labeled proteins into IMVs was carried out by in- cubation at 30 °C for the indicated time periods as described previously (34). conceptually analogous to the sequential proteolysis of RseA by fi fi MICROBIOLOGY DegS followed by RseP (8, 9). The periplasmic domain of RseA Subsequently, NaN3 ( nal concentration of 50 mM), NaCl ( nal concentra- tion of 1 M), and zinc acetate (final concentration of 50 μM) were added and contains Gln-rich regions that contribute to the RseP avoidance the incubation continued at 30 °C. Samples were withdrawn at the indicated (27). Also, binding of RseB to the RseA periplasmic domain time points and treated with 5% trichloroacetic acid. Proteins were solubi- might prevent RseP from cleaving the intact RseA (28). In these lized in SDS sample buffer and analyzed by 10–20% tricine gel electropho- ways, removal of the periplasmic domain transforms RseA into resis (Invitrogen). a substrate of RseP. In the case of presecretory proteins, removal of the large mature domains by Lep might facilitate effective Assessment of the in Vivo Cleavage Site of HA-MBP-Bla from malPEG Modi- access of SPs to the recessed intramembrane of RseP fiability of Engineered Cysteines. Experiments of malPEG modification were (29). This regulation might also require elements in the enzyme carried out essentially as described previously (11). See SI Materials and RseP itself. A pair of PDZ domains, in tandem in the periplasmic Methods for details. region of RseP, plays crucial roles in suppressing DegS-independent cleavage of intact RseA (30); it could also have a positive role in the ACKNOWLEDGMENTS. We thank National Bioresource Project E. coli for the Keio strains (JW0940, JW2203, and JW2556). This work was supported by cleavage of DegS-processed RseA (31). Our results pose an in- a Grant-in-Aid from Japan Society for the Promotion of Science (to Y.A.) and teresting question about whether the RseP PDZ domains partici- from the Ministry of Education, Culture, Sports, Science and Technology, pate in regulation of SP degradation. The S2P-mediated cleavage Japan (to S.C. and Y.A.).

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