Defective translocation of a signal sequence mutant in a prlA4 suppressor strain of Escherichia coli


J. Tommassen, Department of Molecular Microbiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Fax: + 31 30 2513655, Tel.: + 31 30 2532999, E-mail:


In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem.269, 5564–5571], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position −10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon.


signal-recognition particle


ribosome-nascent chain


proton-motive force




inverted inner-membrane vesicle




trigger factor


transmembrane segment

Most proteins destined for the periplasm or the outer membrane of Escherichia coli are transported across the inner membrane by the membrane-embedded Sec system, a complex consisting of the SecYEG translocon, the heterotrimer SecDFyajC and the peripheral ATPase SecA [1,2]. Targeting to the translocon is usually mediated by SecB, which interacts with the mature portion of presecretory proteins [3,4]. The SecB–preprotein complex is then targeted to SecA, which in turn binds with high affinity to SecYEG [5,6]. Upon initiation of translocation, SecB is released [7] and the preprotein is translocated through the translocon by an insertion–deinsertion cycle of SecA [8]. At the periplasmic side of the membrane, leader peptidase removes the signal sequence from the precursor, and the mature protein is released into the periplasm [9]. Targeting of inner-membrane proteins is primarily controlled by the signal-recognition particle (SRP) pathway [2,10,11]. The Ecoli SRP consists of a single protein, P48, and a 4.5S RNA, and binds to particularly hydrophobic signal sequences when they emerge from the ribosome [12,13]. The resulting SRP–ribosome-nascent-chain (SRP–RNC) complex is then targeted via FtsY to the inner membrane [14,15]. Upon release of the SRP, the nascent chain inserts into the membrane near the translocase components SecA, SecY, and SecG, indicating that the SecB-targeting and SRP-targeting pathways converge at a common translocon [16].

The components of the Sec system were originally identified by two different genetic approaches. One method implicated the isolation of conditionally lethal mutants with generalized secretion defects. The second approach emanated from the idea that the signal sequence is recognized by components of the export apparatus, and that specific mutations in genes for Sec components would restore the recognition of mutated signal sequences. Indeed, this method resulted in the isolation of extragenic suppressor mutations in prl (protein localization) genes directly involved in protein translocation (prlA alleles of secY, prlD alleles of secA and prlG alleles of secE) [17]. However, the lack of allele specificity of prlA and prlG mutations with respect to the suppression of signal sequence defects and the observation that these prl mutants are able to translocate even proteins without a signal sequence [18–20] argue against the basic idea of the screening method. Studies with a prlA4 suppressor strain revealed that the Sec translocon in this strain facilitates translocation of preproteins with folded domains [21], showed an increased affinity for SecA [22,23], and is composed of subunits that are more loosely associated than in the wild-type strain [24]. Therefore, it has been proposed that prlA mutations cause a general relaxation of the export apparatus [21,24] rather than a specific change that results in bypassing a proofreading mechanism of the Sec machinery [25].

The energy for precursor translocation is supplied by the hydrolysis of ATP by SecA and by the proton-motive force (pmf) [26]. Energy from ATP binding and hydrolysis is probably used to confer conformational changes in the SecA molecule, which lead to a cycle of insertion and deinsertion of SecA into the membrane [8,27] and movement of the precursor across the membrane [28]. The mechanism by which the pmf stimulates the translocation process is less clear. However, recent experiments indicate that the insertion of the signal sequence in a transmembrane orientation is stimulated by the pmf [29,30]. In our laboratory, we use the SecB-dependent outer-membrane protein PhoE [3,31] as a model protein to study protein transport. In previous studies, we showed that a single amino-acid substitution, G-10L (the residue at position −1 precedes the signal-peptidase cleavage site), in the hydrophobic core of the signal sequence of PhoE relieved the pmf dependency of protein translocation [29] and shunts the precursor via the SRP pathway to the Sec machinery [32]. Other experiments revealed that the prlA4 mutation in secY reduced the pmf dependency of protein translocation of wild-type precursors [21]. The initial goal of the present study was to investigate whether the prlA4 mutation is able to suppress the pmf dependency of the translocation of (G-10L)prePhoE even further. Instead, we found a strong accumulation of the (G-10L)PhoE precursor in the prlA4 mutant strain, and the step that was blocked in the biogenesis pathway was identified.

Materials and Methods

Reagents and biochemicals

Restriction enzymes were purchased from either Boehringer Mannheim or Pharmacia. MEGAshortscript T7 transcription kit was from Ambion, and [35S]methionine and Tran35S-label were from Amersham International. The cross-linker disuccinimidyl-suberate (DSS) and Super Signal West Pico Chemiluminescent Substrate were from Pierce.

Bacterial strains and plasmids

The Ecoli strains and plasmids used in this study are listed in Table 1. To obtain a prlA4 derivative of strain CE1224, a Tn10 marker was introduced close to the prlA4 allele in strain NT1004 by P1 transduction using strain CAG12071 as the donor and selection for tetracycline-resistant transductants. Subsequently, the resulting strain, CE1510, was used as the donor in a P1 transduction experiment with CE1224 as the recipient. To confirm the prlA4 phenotype, tetracycline-resistant transductants were transformed with plasmid pNN6 and tested in pulse-labeling experiments for suppression of the secretion defect of (G-10R)prePhoE, which contains a charged residue in the hydrophobic core of the signal sequence. Tetracycline-resistant prlA+ and prlA4 transductants were designated CE1511 and CE1512, respectively. CE1518-1521 strains were obtained by P1 transduction using strains AF111, ROA7, ROA11 and ROA202, respectively, as recipients and strain POP1730 as the donor.

Table 1. Bacterial strains and plasmids used in this study. Camr and Ampr indicate resistance to chloramphenicol and ampicillin, respectively.
DesignationRelevant characteristicsDescription/reference
 CE1224F, thr leuΔ(proA-proB-phoE-gpt) his thi argE lacY galK xyl rpsL supE ompR[57]
 MC4100F, ΔlacU169 araD139 rpsL thi relA[58]
 NT1060MC4100 ptsF25 deoC1 lamBΔ60T.J. Silhavy (pers. comm.)
 NT1004NT1060 prlA4T.J. Silhavy (pers. comm.)
 CE1510NT1004 zhd-3082::Tn10This study
 CE1511CE1224 zhd-3082::Tn10This study
 CE1512CE1224 prlA4 zhd-3082::Tn10This study
 POP1730ompR::Tn10 ΔmalBK. Bauer
 AF111MC4100 lamB14D[60]
 ROA7AF111 prlA7T.J. Silhavy
 ROA11AF111 prlA11T.J. Silhavy
 ROA202AF111 prlA202T.J. Silhavy
 CE1518AF111 ompR::Tn10This study
 CE1519ROA7 ompR::Tn10This study
 CE1520ROA11 ompR::Tn10This study
 CE1521ROA202 ompR::Tn10This study
 CU165MC4100 secY40 zhd33::tet[44]
 pLep-WTAmpr, lepB behind ara promoter[40]
 pJP29Camr, wild-type phoE[33]
 pNN6pJP29 derivative encoding (G-10R)prePhoE[29]
 pNN8pJP29 derivative encoding (G-10L)prePhoE[29]
 pNN100Ampr, phoE gene behind tac promoter[61]
 pHA106pNN100 derivative encoding (G-10R)prePhoEThis study
 pHA108pNN100 derivative encoding (G-10L)prePhoEThis study
 pC4Meth94PhoEAmpr, encodes truncated 94PhoE[12]
 pC4Meth101FtsQ-WTAmpr, encodes truncated 101FtsQ[12]
 pC4Meth(G-10L)94PhoEpC4Meth94PhoE derivative encoding (G-10L) mutant PhoE[32]

For the construction of plasmids pHA106 and pHA108, encoding the G-10R and G-10L mutant PhoE, respectively, under control of the tac promoter, a PstI/BamHI fragment from pNN100 containing the signal-sequence-encoding part of phoE was replaced by the corresponding fragment of pNN6 and pNN8, respectively.

Pulse-labeling experiments

Strains carrying plasmid pJP29 or derivatives were grown under phosphate limitation at 30 °C to induce PhoE expression [33]. Strains, carrying plasmids with phoE under tac promoter control, were grown for 4 h at 30 °C in synthetic minimal medium [34] supplemented with 100 µg·mL−1 ampicillin and 0.4% glucose. PhoE expression was induced under these conditions with 1 mm isopropyl β-d-thiogalactopyranoside for 1 h. Cells were pulse-labeled for 45 s with Tran35S-label followed either by a chase period with an excess of nonradioactive methionine/cysteine or by chilling on ice. Subsequently, proteins were precipitated with 5% (w/v) trichloroacetic acid, followed by immunoprecipitation with a polyclonal PhoE-specific antiserum [35]. The precipitated proteins were separated by SDS/PAGE [36] and visualized by autoradiography. Radiolabeled proteins were quantified using the Imagequant software (Molecular Dynamics) after scanning of the autoradiogram.

In vitro transcription, translation, targeting and cross-linking analysis

To generate truncated mRNA, plasmids encoding truncated nascent chains of FtsQ or (G-10L)prePhoE (Table 1) were linearized and transcribed as described [12]. The resulting mRNAs, encoding (G-10L)94PhoE and 101FtsQ, were translated in vitro in a lysate of strain MC4100 as described [12,37] to produce RNC complexes. To allow SRP–RNC complex formation, 350 nm reconstituted SRP was added to the translation reaction [16]. After 5 min of incubation at 25 °C, samples were chilled on ice, and the resulting SRP–RNC complexes were purified from the translation mixture by centrifugation through a high-salt sucrose cushion [38] and resuspended in RN buffer (100 mm potassium acetate, 5 mm magnesium acetate, 2.5 mm Hepes/KOH, pH 7.9). Inverted inner-membrane vesicles (IMVs) were isolated as described [37]. Targeting reactions were performed as described previously [16] by incubating purified SRP–RNC complexes together with 1 mm FtsY, 50 µm GTP, 50 µm ATP, and IMVs (1.25 mg·mL−1 protein). Cross-linking was induced with 1 mm DSS for 10 min at 25 °C and quenched at 0 °C by adding 0.1 vol. quenching buffer (1 m glycine/100 mm NaHCO3, pH 8.5). To separate integral membrane from soluble and peripheral cross-linked complexes, samples were treated with 0.18 m Na2CO3 (pH 11.3) for 15 min on ice. The membrane fractions containing integral-membrane proteins were pelleted by ultracentrifugation (10 min, 110 000 g) and resuspended in RN buffer. Supernatant and pellet fractions were precipitated with 10% (w/v) trichloroacetic acid, washed with cold acetone, and resuspended in RN buffer. Samples were immunoprecipitated as described [39] or mixed directly with 2 × SDS/PAGE gel loading buffer before electrophoresis. Samples were analyzed on 12% or 4–15% gradient SDS/polyacrylamide gels. Radiolabeled proteins were visualized by phosphorimaging using a PhosphorImager 473 (Molecular Dynamics).

In vivo membrane-targeting assay

Membrane targeting of leader peptidase was studied in vivo essentially as described [40]. Briefly, cells containing pLep-WT, encoding leader peptidase, were induced with 0.2% arabinose and labeled in the mid-exponential phase with Tran35S-label. After spheroplasting, the cells were treated with proteinase K to degrade translocated proteins, followed by immunoprecipitation [35] with polyclonal antibodies directed against leader peptidase and analysis by SDS/PAGE and autoradiography.

Proteinase K-accessibility experiments

Cells of prlA4 mutant strain NT1004, carrying plasmid pNN8, were grown under phosphate limitation to induce PhoE expression [33] before pulse-labeling with Tran35S-label for 60 s. Directly after the pulse, an excess of nonradioactive methionine and cysteine was added, and the cells were collected by centrifugation (2 min, 16 000 g). For spheroplasting, cells were resuspended in ice-cold buffer A [40% (w/v) sucrose, 1.5 mm EDTA, 33 mm Tris/HCl (pH 8.0)] and incubated with lysozyme (final concentration 5 µg·mL−1). After 10 min incubation on ice, incubation was continued at 37 °C for 10 min followed by addition of 10 mm MgCl2. Aliquots of the spheroplast suspension were incubated on ice for 30 min in the presence or absence of proteinase K (final concentration 50 µg·mL−1). Subsequently, 2 mm phenylmethanesulfonyl fluoride was added to the cell suspension, and incubation was continued for 5 min on ice. Proteins were precipitated with 5% (w/v) trichloroacetic acid and analyzed by SDS/PAGE and autoradiography.

Western immunoblot analysis

Total cellular proteins were solubilized in sample buffer for 10 min at 100 °C, followed by separation by SDS/PAGE on 15% polyacrylamide gels. After transfer of proteins to nitrocellulose filters (0.45 µm; Schleicher and Schuell) using a Mini Trans-Blot Cell (Bio-Rad Laboratories), the blots were incubated with antibodies directed against SecB [41] and developed by chemiluminescence according to the manufacturer's (Pierce) recommendations.


Accumulation of (G-10L)prePhoE in a prlA4 mutant strain

We have previously shown that the prlA4 mutation in secY reduced the pmf dependency of protein translocation [21]. Similarly, a single amino-acid substitution, replacing the helix-breaking glycine at position −10 in the hydrophobic core of the PhoE signal sequence by leucine (G-10L), relieved the pmf dependency of PhoE protein translocation [29]. To investigate whether the reported effects are additive, we studied the in vivo translocation kinetics of wild-type and mutant PhoE in prlA4 mutant strain CE1512, using processing as a criterion for translocation. The prlA4 phenotype of the strain was confirmed by studying the processing kinetics of (G-10R)prePhoE. The translocation of this mutant precursor, which contains a charged residue in the hydrophobic core of the signal sequence, was severely hampered in the wild-type strain and considerably improved in the prlA4 mutant (Fig. 1A). Directly after the pulse, the mature form of the G-10R mutant was barely detectable in the wild-type strain, whereas the majority of the protein was already processed in the prlA4 mutant. Processing was completed in the prlA4 cells after a 2-min chase period, whereas in the wild-type cells, at this time point, still about 50% of the synthesized (G-10R)prePhoE was unprocessed. Interestingly, whereas the (G-10L) mutant precursor was efficiently processed in wild-type cells, it accumulated abundantly in the prlA4 suppressor strain, indicating that its translocation across the inner membrane was affected by the prlA4 mutation. Thus, whereas the prlA4 mutation has been isolated as a strong suppressor of signal sequence defects, it seemed to have an adverse effect on the translocation of the G-10L PhoE precursor.

Figure 1.

Processing kinetics of prePhoE and mutant derivatives in wild-type and prlA mutant strains. (A) Cells of prlA+ strain CE1511 (prlA+) or prlA4 mutant CE1512, carrying plasmids encoding wild-type PhoE (WT) or mutant derivatives with G-10R or G-10L substititutions in the signal sequence, were grown under phosphate limitation to express PhoE. The cells were pulse-labeled at 30 °C for 45 s with Tran35S-label and chased with unlabeled methionine/cysteine for the indicated periods. The proteins were analyzed, after immunoprecipitation with antibodies against PhoE, by SDS/PAGE and autoradiography. The precursor and mature forms of PhoE are indicated by p and m, respectively. (B) Cells of strains CE1518 (prlA+), CE1519 (prlA7), CE1520 (prlA11) and CE1521 (prlA202), carrying plasmids encoding wild-type PhoE (WT) or mutant derivatives under control of the tac promoter, were grown in synthetic minimal medium at 30 °C until the mid-exponential phase. After 1 h induction with isopropyl β-d-thiogalactoside, aliquots of each culture were pulse-labeled with Tran35S-label for 45 s, followed by the addition of an equal volume of ice-cold 10% trichloroacetic acid. Radiolabeled proteins were subsequently analyzed as described under (A). G, R and L indicate PhoE proteins with a Gly, Arg and Leu at position −10 in the signal sequence (− 10), respectively.

To test whether the (G-10L)prePhoE accumulation is specific for the prlA4 allele, we determined the effect of several other prlA suppressor mutations with the amino-acid substitutions being located in distinct topological domains of SecY [25]. Again, suppression of the translocation defect of (G-10R)prePhoE was used as a control for the suppressor phenotype (Fig. 1B). Quantification of the results revealed that 39% of the total amount of radiolabeled G-10R mutant PhoE was processed directly after the pulse in the wild-type strain. In the prlA7 and prlA202 suppressor strains, processing was improved and the amount of mature PhoE increased to 50% and 60%, respectively, of the total amount of PhoE synthesized during the pulse. The prlA11 suppressor increased the amount of PhoE only by 2% compared with wild-type cells. Although the prlA7 and prlA202 alleles tested improved the processing of (G-10R)prePhoE, precursor accumulation of (G-10L)prePhoE was not observed in these prlA suppressors. In conclusion, these data suggest that (G-10L)prePhoE accumulation is specific for the prlA4 allele.

Targeting of G-10L nascent PhoE to the PrlA4 Sec translocon

Whereas wild-type PhoE is targeted to the Sec translocon by SecB [3,31], targeting of (G-10L)prePhoE is mediated by SRP [32]. Therefore, the accumulation of (G-10L)prePhoE in the prlA4 mutant may result from a defect in SRP-mediated targeting to the mutant translocon. To study this possibility, we examined the targeting of (G-10L)prePhoE nascent chains to SecY in vitro in cross-linking experiments. After translation, RNCs of (G-10L)94PhoE polypeptides were saturated with reconstituted SRP. The SRP–RNC complexes were purified and incubated with IMVs, derived from either a wild-type or a prlA4 mutant strain to allow targeting. After cross-linking with the bifunctional cross-linking reagent DSS, peripheral and soluble cross-linked complexes were separated from integral-membrane cross-linked complexes by Na2CO3 extraction, and the complexes were analyzed by SDS/PAGE (Fig. 2A). In the Na2CO3 pellet, at least two (G-10L)94PhoE cross-linked complexes, one at ≈ 110 kDa and one at ≈ 46 kDa, could be detected with both wild-type and prlA4 IMVs (Fig. 2A, lanes 4 and 5). In both cases, the 110-kDa complex could be immunoprecipitated with antiserum directed against SecA, indicating that it is a complex of the radiolabeled (G-10L)94PhoE and SecA (Fig. 2B, lanes 1 and 3). In addition, cross-linking adducts of ≈ 220 kDa and ≈ 40 kDa were also immunoprecipitated from the Na2CO3 pellet with SecA antiserum. We assume that the ≈ 220-kDa product corresponds to cross-linked complexes between (G-10L)94PhoE and the dimeric form of SecA. The ≈ 40-kDa product in the Na2CO3 pellet probably contains proteolytic fragments of the SecA cross-linked products, which is in agreement with earlier reports [42]. The fuzzy ≈ 46-kDa product (Fig. 2A, lanes 4 and 5) was immunoprecipitated with anti-SecY serum (Fig. 2B, lanes 2 and 4), demonstrating that the (G-10L)94PhoE nascent chains are targeted to the Sec translocon in the prlA4 IMVs as well as in wild-type IMVs. The small difference in the electrophoretic mobilities of the PrlA4-(G-10L)94PhoE adduct and SecY-(G-10L)94PhoE adduct (Fig. 2A, lanes 4 and 5) probably results from the amino-acid substitutions in the mutant PrlA4 protein.

Figure 2.

Targeting of SRP–RNCs to the prlA4 mutant Sec translocon.[35S]Methionine-labeled (G-10L)94PhoE or 101FtsQ was incubated with 350 nm reconstituted SRP. The SRP–RNCs were subsequently purified and incubated with NT1060 (prlA+) and NT1004 (prlA4) IMVs. The cross-linker DSS was used to analyze SRP–RNC interactions. After quenching, peripherally bound and soluble proteins were separated from the inner membranes by carbonate extraction. Samples were either directly (A) or after immunoprecipitation with the indicated antisera (B and C) subjected to SDS/PAGE, and cross-linked complexes were visualized with a PhosphorImager. The positions of molecular mass marker proteins (MW) are indicated on the right. Arrowheads indicate relevant cross-linked complexes.

In the Na2CO3 supernatant, at least three major cross-linking adducts, of apparent molecular mass ≈ 110, ≈ 65 and ≈ 55 kDa, could be detected, both when wild-type and when prlA4 IMVs were present (Fig. 2A, lanes 8 and 9). Immunoprecipitation revealed that these adducts represent cross-linking to SecA (data not shown), trigger factor (TF) and P48 (Fig. 2C), respectively, demonstrating that (G-10L)94PhoE interacts with P48 and TF in both cases. As described in the accompanying paper [32], several additional low-molecular-mass cross-linking adducts (< 30 kDa) were revealed (Fig. 2A, lanes 8 and 9), but the identity of these complexes is unknown. To investigate whether the cross-linking adducts are similar to those of other substrates of the SRP pathway, FtsQ was used as a model substrate. This class II membrane protein, with a short N-terminal cytoplasmic tail [43], was synthesized as a slightly longer nascent chain (101 residues) than (G-10L)94PhoE to expose properly its signal-anchor domain. Indeed, 101FtsQ interacted properly with both SecY and SecA (Fig. 2A, lanes 13 and 14) as demonstrated by immunoprecipitations with anti-SecY and anti-SecA serum, respectively (Fig. 2B, lanes 5–8). In conclusion, the results show that the targeting of the (G-10L)94PhoE nascent chains and other SRP substrates to the SecY protein in the translocon is not defective in the prlA4 mutant.

(G-10L)prePhoE is inefficiently translocated in the prlA4 mutant

Whereas the targeting of the (G-10L)prePhoE and other SRP substrates to the translocon is apparently unaffected in the prlA4 mutant, their subsequent insertion into the mutant translocon might be impaired. To test this possibility, protease-accessibility experiments were conducted after pulse-labeling of cells expressing the G-10L mutant protein (Fig. 3). Indeed, the precursor of G-10L PhoE that accumulated in the prlA4 mutant was not sensitive to proteinase K after spheroplasting of the cells (Fig. 3, lane 2), whereas mature PhoE, which is translocated, was degraded. These results show that the precursor of (G-10L)PhoE, although correctly targeted to the translocon, is inefficiently translocated across the inner membrane in a prlA4 mutant.

Figure 3.

Proteinase K accessibility of (G-10L)prePhoE in prlA4 mutant cells. NT1004 (prlA4) cells, expressing (G-10L)prePhoE from plasmid pNN8, were pulse-labeled for 60 s with Tran35S-label, followed by the addition of an excess of methionine/cysteine. After spheroplasting, the cells were incubated in the absence or presence of proteinase K (PK). Subsequently, the undigested proteins were precipitated with trichloroacetic acid and analyzed by SDS/PAGE and autoradiography. The precursor and mature forms of PhoE are indicated by p and m, respectively.

To investigate whether membrane insertion of SRP substrates is also defective in a prlA4 mutant, we examined the insertion kinetics of leader peptidase [40]. Directly after pulse-labeling, leader peptidase already appeared to be accessible to proteinase K in spheroplasts of both the wild-type and the prlA4 mutant strain (Fig. 4A). The intactness of the spheroplasts was confirmed by the inaccessibility to proteinase K of SecB, which was only degraded after solubilization of the spheroplasts with Triton X-100 (Fig. 4B). These results indicate that insertion of the SRP substrate leader peptidase into the Sec translocon is not affected in the prlA4 mutant.

Figure 4.

In vivo membrane insertion of leader peptidase in wild-type and prlA4 mutant cells. (A) Cells of strain NT1060 (prlA+) or its prlA4 derivative NT1004, carrying plasmid pLep-WT, were grown in synthetic minimal medium to mid-exponential phase at 30 °C. Expression of leader peptidase was induced for 5 min by the addition of 0.2% arabinose. Subsequently, cells were pulse-labeled for 15 s with Tran35S-label and chased with unlabeled methionine/cysteine for the indicated periods. Insertion of leader peptidase was assessed by analyzing its protease accessibility after spheroplasting of the cells. The proteins were analyzed after immunoprecipitation with anti-Lep antibodies by SDS/PAGE and autoradiography. (B) As a control for membrane intactness, the spheroplasts were treated with proteinase K (PK), without or with solubilization by Triton X-100 (TX-100) and analyzed by immunoblotting using antiserum directed against SecB.

Processing of (G-10L)prePhoE is impaired in secY40 mutant cells

The results presented so far indicate that the prlA4 mutation has distinct effects on the translocation kinetics of a SecB-targeted and an SRP-targeted precursor, i.e. wild-type and G-10L mutant PhoE, respectively. A possible explanation is that these substrates are targeted to different domains of SecYEG. A cold-sensitive secY40 mutant has previously been described that is impaired in inner-membrane protein insertion, whereas protein export is unaffected [44]. In addition, this result suggested that different regions of SecY may be involved in protein export and membrane protein insertion. To test whether the secY40 mutation affects the translocation of wild-type and (G-10L)prePhoE differently, pulse–chase experiments were conducted. Indeed, even at the permissive temperature, the (G-10L)prePhoE was only slowly processed in the secY40 mutant, whereas wild-type prePhoE was completely processed within 30 s after the pulse (Fig. 5). These results are consistent with the hypothesis that wild-type and G-10L mutant prePhoE are targeted to different domains of the SecYEG translocon.

Figure 5.

Processing of (G-10L)prePhoE is impaired in secY40 cells. Cells of strain CU165 (secY40) carrying plasmid pJP29 encoding wild-type PhoE (WT) or pNN8 encoding (G-10L)prePhoE, were grown under phosphate limitation to express PhoE. The cells were pulse-labeled at 37 °C for 45 s with Tran35S-label and chased with unlabeled methionine/cysteine for the indicated periods. Radiolabeled proteins were analyzed by SDS/PAGE and autoradiography. The precursor and mature forms of PhoE are indicated by p and m, respectively.


Signal sequences often contain an α-helix breaker in the hydrophobic core [45]. We have previously shown that substitution of the helix breaker in the signal sequence of PhoE by a helix-promoting residue (e.g. G-10L) relieved the pmf dependency of the translocation of this precursor [29]. Furthermore, we showed that such substitutions resulted in the re-routing of this SecB substrate to the SRP-targeting pathway [32]. In the present study, we show that (G-10L)prePhoE accumulates in its precursor form when expressed in a prlA4 mutant strain and that its translocation across the inner membrane is impaired.

The results described here for (G-10L)prePhoE are reminiscent of those reported previously for staphylokinase (Sak), a secreted protein from the Gram-positive bacterium Staphylococcus aureus, which was efficiently processed and exported to the periplasm in wild-type Ecoli cells, but accumulated in its precursor form when expressed in a prlA4 mutant of Ecoli[46]. Sequence examination suggests the presence of an unusually long α-helix in the core region of the Sak signal sequence as is the case in the (G-10L)prePhoE signal sequence. The export defect of Sak in the prlA4 mutant was suppressed by a small four-amino-acid deletion or by amino-acid substitutions introducing a strong helix breaker, such as glycine, into the α-helical core region of the signal sequence [46]. Similarly, streptokinase, an extracellular protein of streptococcal strains, is also blocked from being secreted in Ecoli prlA4 mutant cells [47], presumably because of the long α-helix in its signal sequence.

The prlA4 allele actually contains two missense mutations in the secY gene, resulting in the amino-acid substitutions F286Y and I408N in transmembrane segments (TMS) 7 and 10, respectively [48]. The mutation in TMS 10 is responsible for the suppressor phenotype and enables the translocation of preproteins with a defective or completely missing signal sequence [20,25,49]. Furthermore, this mutation was reported to result in a looser association between the subunits of the SecYEG translocon [24] and in increased affinity of SecA for the SecY protein [23]. The mutation in TMS 7 was probably acquired as a secondary mutation which restored the stability while conserving the flexibility of the system. Further, it was shown that the mutation in TMS 7 (F286Y) is responsible for the observed export defect of Sak [46,48,50], suggesting that this defect is not related to the suppressor phenotype. Consistently, we did not observe a processing defect when (G-10L)prePhoE was expressed in other prlA suppressor strains, i.e. prlA7 (A277E in TMS 7 and L407R in TMS 10), prlA11 (V407R in TMS 10 and V411G in TMS 10), and prlA202 (I287S in TMS 7). Therefore, we speculate that the export defect of (G-10L)prePhoE, like that of Sak, is caused by the secondary mutation in TMS 7 of SecY.

Recent studies have indicated that the SecYEG complex facilitates the insertion of inner-membrane proteins in addition to catalyzing protein translocation [51,52]. Membrane protein insertion and protein translocation are two distinct processes that are likely to impose different functional requirements on the translocase. The mechanism by which the Sec complex mediates both of these functions is still unclear. Available evidence indicates that the translocon forms an aqueous channel that permits the translocation of polypeptides into the periplasm [53,54]. However, it can also open laterally to allow the exit of transmembrane regions into the lipid bilayer [55]. Possibly, the passage of exported proteins and the insertion of integral membrane proteins are facilitated by distinct regions of the translocon. Indeed, studies in eukaryotes indicated that signal sequences of secreted proteins and signal-anchor domains of membrane proteins are positioned differently in the Sec61p translocon [56]. Recently, a secY40 mutant of Ecoli (carrying an A363S substitution in cytoplasmic domain 5 of SecY) was shown to be defective in inner-membrane protein insertion, whereas protein export was unaffected by the mutation [44]. Our data show that the translocation of the SRP substrate (G-10L)prePhoE is affected by the secY40 mutation as well, whereas the SecB substrate wild-type PhoE is not, indicating that these highly related precursors are differently processed at the SecYEG translocon. Furthermore, the (G-10L)prePhoE accumulation in prlA4 cells is caused by inefficient translocation, whereas wild-type prePhoE is correctly translocated. However, the membrane insertion of another SRP substrate, leader peptidase, was not affected by the prlA4 mutation. To explain our results, we propose that SRP and SecB substrates are targeted to different domains of the translocon. The cytoplasmic domain 5, where the secY40 mutation is located, is involved in the docking of SRP substrates. Also after docking at the translocon, SRP substrates and SecB substrates are differently processed by the translocon. In the case of SRP substrates, the translocon opens laterally to allow the insertion of integral-membrane proteins. However, when the hydrophobic α-helix is too short to span the inner membrane, the SRP substrate may be transferred to the translocation pathway, which is normally used by SecB substrates. This transfer appears to be defective in the prlA4 mutant.


We would like to thank Malene Urbanus for her efforts with the cross-linking experiments. Furthermore, we would like to thank Elaine Eppens and Margot Koster for helpful discussions and interest in the work, William Wickner, Annemieke van Dalen and Arnold Driessen for providing antisera, and Katharina Bauer, Ann Flower, Chris Harris and Tom Silhavy for their gifts of strains. This work was supported by EU grant HPRN-CT-2000-00075 from the European Community.