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Export of autotransported proteins proceeds through an oligomeric ring shaped by C-terminal domains

2002; Springer Nature; Volume: 21; Issue: 9 Linguagem: Inglês

10.1093/emboj/21.9.2122

1460-2075

Esteban Veiga,

Bacteriophages and microbial interactions

Article1 May 2002free access Export of autotransported proteins proceeds through an oligomeric ring shaped by C-terminal domains Esteban Veiga Esteban Veiga Departmento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Madrid, 28049 Spain Search for more papers by this author Etsuko Sugawara Etsuko Sugawara Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720-3206 USA Search for more papers by this author Hiroshi Nikaido Hiroshi Nikaido Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720-3206 USA Search for more papers by this author Víctor de Lorenzo Víctor de Lorenzo Departmento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Madrid, 28049 Spain Search for more papers by this author Luis Angel Fernández Corresponding Author Luis Angel Fernández Departmento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Madrid, 28049 Spain Search for more papers by this author Esteban Veiga Esteban Veiga Departmento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Madrid, 28049 Spain Search for more papers by this author Etsuko Sugawara Etsuko Sugawara Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720-3206 USA Search for more papers by this author Hiroshi Nikaido Hiroshi Nikaido Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720-3206 USA Search for more papers by this author Víctor de Lorenzo Víctor de Lorenzo Departmento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Madrid, 28049 Spain Search for more papers by this author Luis Angel Fernández Corresponding Author Luis Angel Fernández Departmento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Madrid, 28049 Spain Search for more papers by this author Author Information Esteban Veiga1, Etsuko Sugawara2, Hiroshi Nikaido2, Víctor de Lorenzo1 and Luis Angel Fernández 1 1Departmento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Madrid, 28049 Spain 2Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720-3206 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:2122-2131https://doi.org/10.1093/emboj/21.9.2122 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info An investigation was made into the oligomerization, the ability to form pores and the secretion-related properties of the 45 kDa C-terminal domain of the IgA protease (C-IgAP) from Neisseria gonorrhoeae. This protease is the best studied example of the autotransporters (ATs), a large family of exoproteins from Gram-negative bacteria that includes numerous virulence factors from human pathogens. These proteins contain an N-terminal passenger domain that em bodies the secreted polypeptide, while the C-domain inserts into the outer membrane (OM) and trans locates the linked N-module into the extracellular medium. Here we report that purified C-IgAP forms an oligomeric complex of ∼500 kDa with a ring-like structure containing a central cavity of ∼2 nm diameter that is the conduit for the export of the N-domains. These data overcome the previous model for ATs, which postulated the passage of the N-module through the hydrophilic channel of the β-barrel of each monomeric C-domain. Our results advocate a secretion mechanism not unlike other bacterial export systems, such as the secretins or fimbrial ushers, which rely on multimeric complexes assembled in the OM. Introduction Proteins secreted by Gram-negative bacteria must cross the lipid bilayers of the inner (IM) and outer (OM) membranes as well as the periplasmic space containing the peptidoglycan layer (Nikaido, 1996; Duong et al., 1997; Bernstein, 2000). To achieve this formidable task, different secretion systems have evolved (Lory, 1998; Thanassi and Hultgren, 2000). Some involve the initial translocation of a protein precursor with an N-terminal signal peptide into the periplasmic space (e.g. type II and chaperone/usher pathways) (Russel, 1998; Thanassi et al., 1998a; Soto and Hultgren, 1999; Sandkvist, 2001). The secreted polypeptide, either alone or in association with a periplasmic chaperone, can then be recognized by an OM protein complex that promotes its final translocation to the extracellular medium. Alternatively, some polypeptides are secreted without a periplasmic intermediate by passing directly from the cytoplasm into the extracellular medium through a hydrophilic protein channel connecting the IM and OM (e.g. type I and type III pathways) (Blight and Holland, 1994; Thanabalu et al., 1998; Cheng and Scheneewind, 2000; Buchanan, 2001; Fernández and de Lorenzo, 2001; Plano et al., 2001). A common feature of all these pathways is the participation of several gene products in the assembly of a secretion apparatus, usually some 3–20 polypeptides with at least one being an OM oligomer containing a central hydrophilic pore (Russel, 1998; Stathopoulos et al., 2000). Autotransporters (ATs) are a distinct family of secreted proteins which contain all the necessary elements for translocation across the OM within their own polypeptide sequences (Henderson et al., 1998, 2000). Members of this family include virulence factors of important human pathogens, such as the IgA1 proteases from Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus influenzae (Pohlner et al., 1987; Lomholt et al., 1995), the actin polymerization factor IcsA from Shigella flexneri (Suzuki et al., 1995), the AIDA-I adhesin from pathogenic Escherichia coli (Benz and Schmidt, 1992; Suhr et al., 1996), the serum-resistant factor BrkA from Bordetella pertussis (Fernandez and Weiss, 1994) and the cytotoxin VacA from Helicobacter pylori (Schmitt and Haas, 1994) among others (Henderson and Nataro, 2001). The fundamental mechanism of AT secretion was outlined by studies of the IgA1 protease (IgAP) from N.gonorroheae (Pohlner et al., 1987). The iga gene encodes a large precursor (∼170 kDa) in which the mature protease (∼106 kDa) is flanked by an N-terminal signal peptide directing the initial export into the periplasm, and an ∼45 kDa (C-IgAP) C-terminal domain starting at Val1124 that inserts into the OM and which is required for translocation of the protease domain toward the bacterial surface (Figure 1A). During this step, autoproteolytic processing releases the mature protease into the extracellular medium. These studies showed that C-IgAP could translocate a heterologous passenger domain lacking disulfide bonds towards the bacterial surface (i.e. the 15 kDa cholera toxin B subunit) (Klauser et al., 1990, 1992). Protection experiments against externally added proteases indicated that the last ∼30 kDa of C-IgAP, the so-called β-core, are embedded in the OM in vivo (Klauser et al., 1993). Predictions of the secondary structure of C-IgAP suggested that this β-core may contain 15 amphipathic β-strands that could fold as a β-barrel similar to those found in outer membrane proteins (OMPs) (e.g. OmpA, OmpF, PhoE, LamB, FhuA, FepA and TolC; for reviews see Koebnik et al., 2000; Tamm et al., 2001). Figure 1.Structural organization of the IgA protease and HEβ hybrid. (A) Schematic representation of the IgA protease precursor (∼170 kDa) from N.gonorroheae showing the position of the N-terminal signal sequence (ss), the secreted protease module (∼120 kDa) and the transporter C-domain (∼45 kDa; C-IgAP). The ∼30 kDa β-core region within C-IgAP is also labeled. (B) Organization of the relevant insert of plasmid pHEβ encoding the HEβ hybrid under the control of the IPTG-inducible lac promoter (plac). The heb gene chimera is an in-frame fusion of DNA segments encoding the pelB N-terminal signal sequence (ss), polyhistidine (6×his) and E-tag peptides, and the C-IgAP transporter domain. The site of cleavage of the signal peptidase (just before the His6 peptide) is indicated by an arrowhead. (C) Representation of topology of HEβ in the OM showing the His6 N-passenger domain exposed to the extracellular space (Out) and the C-terminus toward the periplasm. The putative β-barrel of HEβ is depicted as a cylinder embedded in the OM. Download figure Download PowerPoint The above led to the proposal of an elegant model for AT secretion in which the β-barrel of a monomeric C-domain contained a hydrophilic channel through which an unfolded N-domain could be translocated (Klauser et al., 1990, 1992). However, this model has received no experimental support. Indeed, it has been challenged recently in view of new data showing that homologous and heterologous N-domains can be translocated after folding by the action of periplasmic chaperones (Veiga et al., 1999; Valls et al., 2000; Brandon and Goldberg, 2001). These results raise the question of the actual size and nature of the pore used for secretion in ATs. The present work investigates the structural and functional properties of the transporter C-IgAP domain from N.gonorroheae. The results show that C-IgAP assembles as a ring-like oligomeric complex of ∼500 kDa with a central hydrophilic pore of ∼2 nm diameter. This structure is similar to that of the OM complexes found in other secretion systems (e.g. secretins and fimbrial ushers; Thanassi et al., 1998b; Stathopoulos et al., 2000). Furthermore, evidence was obtained indicating that the central pore of the C-IgAP complex is the site used for the secretion of the N-domains. Results Purification of native C-IgAP A gene construct encoding a functional polyhistidine-tagged version of the 45 kDa C-IgAP was generated. This hybrid protein, named HEβ (∼50 kDa), contained an N-terminal signal peptide (pelB) followed by six histidines (His6) and a 12 amino acid peptide (E-tag) fused to C-IgAP (Figure 1B and C). When produced into E.coli, HEβ was localized in the OM fraction, with the His6 and E-tag peptides fully exposed toward the bacterial surface [according to whole-cell enzyme-linked immunosorbent assay (ELISA) and digestion with externally added proteases; data not shown]. HEβ showed strong resistance to denaturation in the presence of 1% SDS unless the temperature was increased to 100°C. This was revealed by the two distinct electrophoretic mobilities for HEβ observed in SDS–PAGE, with migration as a faster protein band if the sample was not boiled (Figure 2A). The heat-modifiable electrophoretic mobility of HEβ is a typical feature of OMPs with β-barrel structure, with the folded conformation (more compact) migrating more quickly in polyacrylamide gels than the denatured form (Schnaitman, 1973; Nikaido and Vaara, 1985). Advantage was taken of this behavior to monitor the folding state of HEβ throughout its purification. Figure 2.Electrophoretic mobility and purification of HEβ. (A) Western blot developed with anti-E-tag mAb–POD of whole-cell protein extracts from IPTG-induced E.coli UT5600 harboring pHEβ. Before loading onto the 10% polyacrylamide gel, the samples were resuspended in denaturing SDS–PAGE sample buffer and heated for 10 min at the indicated temperatures (25, 42, 50, 65 and 100°C). The faster mobility band of HEβ (f) corresponds to the folded conformation of the hybrid. When unfolded, HEβ migrates as a slower mobility band (u). (B) A sample of purified HEβ is shown after Coomassie Blue staining of a 10% SDS–polyacrylamide gel. (C) Western blot developed with anti- E-tag mAb–POD of purified HEβ samples treated at 25 (lane 1) or 100°C (lane 2) for 10 min before loading onto a 10% SDS– polyacrylamide gel. (D) The possible presence of contaminating OmpF porin in the purified HEβ was evaluated by western blot developed with a polyclonal serum against trimeric OmpF. Excess purified HEβ (10 μg, lanes 1 and 2) and a sample of purified OmpF (0.1 μg, lanes 3 and 4) as a control were loaded onto a 10% SDS–polyacrylamide gel after heating at 25 (lanes 1 and 3) or 100°C (lanes 2 and 4) for 10 min. Only the trimeric OmpF control (lane 3) was detected, ruling out the presence of OmpF in the purified HEβ sample. Download figure Download PowerPoint To this end, HEβ was produced in E.coli UT5600, a strain lacking the OM protease OmpT (Grodberg and Dunn, 1988). After isolation of the OM fraction from these cells, the OMPs were solubilized in a buffer containing 1% Zwittergent 3–14 and passed through a cobalt-containing resin for immobilized metal affinity chromatography (IMAC). This detergent was instrumental in solubilizing HEβ without affecting its heat-modifiable electrophoretic mobility, even after extended incubations. Solubilized HEβ bound specifically to the IMAC column and was eluted in a buffer containing imidazole and 0.1% Zwittergent 3–14. Finally, the imidazole was removed by dialysis and HEβ was concentrated (see Materials and methods). This purification procedure gave a protein band corresponding to HEβ after Coomassie Blue staining of a denaturing SDS–polyacrylamide gel (Figure 2B) and western blotting with anti-E-tag monoclonal antibody (mAb) (Figure 2C). The folding of purified HEβ was tested by its heat-modifiable mobility in SDS–PAGE (Figure 2C). In this analysis, it was found that the non-boiled sample of HEβ generated bands of high molecular weight (Figure 2C, lane 1). As an additional purity criterion, western blots were used to show that the HEβ preparation contained no detectable trace of the major E.coli porin, OmpF (Figure 2D). The secondary structure of purified HEβ was investigated using circular dichroism (CD). The CD spectrum obtained using 0.1 mg/ml of HEβ in a buffer containing 0.1% Zwittergent 3–14 is shown in Figure 3. The structural components derived from this spectrum (Perczel et al., 1992) indicate that HEβ contains 30.2% β-sheet conformation, 34.4% α-helix structure, 9.8% turns and 25.6% unordered structure. This analysis is compatible with the presence of 15 amphipathic β-strands (Klauser et al., 1993), which represent 30% of the HEβ sequence, but reveals a rich α-helical content in C-IgAP. In addition, these data confirmed that HEβ remained in a structured state after purification. Figure 3.CD spectrum of HEβ. The CD spectrum of purified HEβ (0.1 mg/ml) was monitored at 22°C in TNZ buffer [20 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.1% (w/v) Zwittergent 3–14]. A minimum of four spectra were accumulated and the contribution of the buffer subtracted. Values of mean residue weight ellipticities (Θ) M.R.W. (degrees × cm2 × dmol−1) are indicated. Download figure Download PowerPoint Biochemical measurement of the pore in C-IgAP The possible presence of a hydrophilic conduit in HEβ was examined using the proteoliposome swelling assay, which has been used to study the pore-forming activity of porins (Nikaido and Rosenberg, 1981; Nikaido et al., 1991; Nikaido, 1994). Briefly, purified HEβ was embedded into multilamellar liposomes suspended in solutions containing an iso-osmotic concentration of sugars of different Mr. If a hydrophilic pore were present in HEβ, the sugars, concomitant with water, would enter into the liposomes, producing their swelling. This can be monitored as a decrease in the OD400. Pore-forming activity was clearly detected in the proteoliposomes containing HEβ and suspended in arabinose solutions (Mr ∼150 Da). Porin activity was directly proportional to the amount of HEβ used (Figure 4A). Addition of 2 nmol of E.coli lipopolysaccharide (LPS) increased the absolute swelling rate of the liposomes containing HEβ by ∼3-fold (e.g. from 0.14 to 0.4 ΔOD400/min at 1.4 μg of HEβ in arabinose). No swelling was noticed in proteoliposomes reconstituted with bovine serum albumin (BSA; negative control), whereas those with OmpF (positive control) had swelling rates identical to those previously reported (Nikaido and Rosenberg, 1981; Nikaido et al., 1991). Figure 4.Pore-forming activity of HEβ. (A) Swelling rates (ΔOD400/min) of proteoliposomes suspended in isosmotic solutions of arabinose and containing the indicated amount of purified HEβ. (B) Swelling rates of proteoliposomes containing HEβ in solutions of sugars with different Mr. The sugars used were arabinose (Ara; 150 Da), glucose (Glu; 180 Da), N-acetylglucosamine (Nag; 221 Da), sucrose (Suc; 342 Da) and raffinose (Raf; 504 Da). The data are shown relative to the swelling in arabinose and are the averages of at least six independent experiments in which a range of amounts of HEβ (from 0.8 to 2.5 μg) was employed. The swelling rate corresponding to 10% of that in arabinose is indicated with a dashed line. (C) The Mr (0.1 Ara) of HEβ (410 Da) was intersected in a plot representing the Mr (0.1 Ara) of OMPs versus their pore size (PhoE, 240 Da and 1.1 nm; OmpC, 236 Da and 1.1 nm; OmpF, 255 Da and 1.2 nm; OmpG, 400 Da and 2.0 nm; PapC, 620 Da and 3 nm) (Nikaido and Rosenberg, 1983; Cowan et al., 1992; Fajardo et al., 1998; Thanassi et al., 1998b). This plot allows an estimation of 2 nm for the size of HEβ. Download figure Download PowerPoint The size of the channel in HEβ was estimated from the swelling rates with sugars of different Mr in the presence or absence of LPS. When these rates were expressed relative to those obtained with arabinose, the sugar with the lowest Mr, it was apparent that the different sugars had identical relative values in either the presence or absence of LPS (Table I). The relative swelling rates were plotted versus the Mr of the sugars employed to obtain the parameter Mr (0.1 Ara) for HEβ of 410 Da (Figure 4B). This parameter, defined as the Mr of a solute that would diffuse at 10% of the rate of arabinose, has been used extensively to calculate the diameter of channels in OMPs (Nikaido et al., 1991). Comparing the Mr (0.1 Ara) of HEβ with that of other OMPs of known pore size, the hydrophilic channel of the C-IgAP was estimated to have a diameter of ∼2 nm (Figure 4C). Table 1. Relative swelling rates of liposomes containing HEβ (%) Sugar Mr LPS − + Arabinose 150 100 100 Glucose 180 60 59 N-acetylglucosamine 221 20 23 Sucrose 342 15 12 Raffinose 504 not tested 5 The data presented are the average of at least three independent experiments in which triplicates of each point were measured.The typical deviation was always <17% of the average values.The rates were normalized to that in arabinose, which was taken as 100%. To confirm the porin activity detected in HEβ, a different C-IgAP hybrid (Corβ) was used that contained a 10 amino acid viral epitope as the N-domain (Veiga et al., 1999). In addition, it was investigated whether the presence of a sizable N-domain—a recombinant single-chain Fv (scFv) antibody with two disulfide bonds (Mr ∼30 kDa) (Veiga et al., 1999)—influenced the pore-forming activity of the C-IgAP. In these experiments, the hybrid proteins Corβ and FvHβ were expressed in E.coli HN705, a strain lacking the OmpC and OmpF porins (Sugawara and Nikaido, 1992). The OM fractions from these cells were isolated by isopycnic centrifugation and identical amounts of total protein of these OM fractions were used in the liposome swelling assays (see Materials and methods). This E.coli strain reproducibly gave rise to low levels of Corβ and FvHβ hybrids, detected in the OM fractions by western blotting with anti-E-tag mAb. For unknown reasons, the amount of FvHβ was always ∼100-fold higher than that of Corβ in E.coli HN705. Despite these facts, the swelling of the liposomes containing Corβ and FvHβ was readily detectable in arabinose and sucrose, whereas the OM fraction from the non-transformed E.coli HN705 strain induced no significant liposome swelling in sucrose (Table II). Since FvHβ was present in greater amounts than Corβ in these OM fractions, and pore-forming activity was directly proportional to the amount of protein added (Figure 4A), it is very likely that the presence of a bulky scFv N-domain blocked most of the porin activity of C-IgAP. In a previous study, we have shown that a small proportion of FvHβ hybrids (∼15%) fully translocate the folded scFv across the OM, while the remaining hybrids (∼85%) have their N-domain within the OM fraction (Veiga et al., 1999). Table 2. Swelling rates of liposomes containing OM fractions Escherichia coli cells Arabinose Sucrose HN705 0.26 U HN705/Corβ 0.4 0.04 HN705/FvHβ 0.47 0.05 Values presented came from at least three different measurements. OM fractions containing 60 μg of total protein were added to liposomes. The typical deviation was always 250 kDa were detected in denaturing SDS–polyacrylamide gels by western blot with anti-E-tag mAb (Figure 5B, lane 1), which probably correspond to a monomer, dimer, trimer and at least one tetramer of HEβ. As expected, these high Mr bands appeared after treatment with DSP and were dissociated with 2-ME (Figure 5B, lanes 2–4). This pattern of cross-linking of HEβ in vivo was confirmed with a different reagent, disuccinimidyl glutarate (DSG, 7.7 Å spacer) (data not shown). The mass of the oligomeric complex formed by HEβ was assessed by size exclusion chromatography. The elution profile of HEβ in a gel filtration column with an exclusion limit of 1500 kDa (Bio-Gel A-1.5m) is provided in Figure 6. The quantity of HEβ in the different fractions was determined by western blot with anti-E-tag mAb. Proteins of known mass were separated along with HEβ as size markers for generating a standard curve. HEβ eluted as a single peak with an apparent mass of ∼500 kDa. Remarkably, HEβ was not detected in the fractions corresponding to the mass of the monomer (50 kDa). This result was confirmed with three independent preparations of purified HEβ and employing a different chromatographic medium (Macro-Prep SE 1000/40). Figure 6.Size exclusion chromatography of purified HEβ. The elution profile in a Bio-Gel A column of HEβ and proteins of known Mr as standards [thyroglobulin (thy; 670 000 Da), bovine γ-globulin (ggb; 158 000 Da), chicken ovalbumin (ova; 44 000 Da), equine myoglobin (myo; 17 000 Da) and vitamin B-12 (b-12; 1350 Da)] is shown. Elution of the Mr standards was monitored by UV absorption at 280 nm (open circles), whereas HEβ was detected by western blotting with anti-E-tag mAb–POD. Download figure Download PowerPoint The large size estimated for the HEβ complex raised the possibility that it might be visible by electron microscopy. Thus, samples containing purified HEβ were negatively stained with ammonium molybdate (Figure 7A) or uranyl acetate (Figure 7B) and viewed with an electron microscope. In the unprocessed micrographs obtained with both negative stains, HEβ appeared as ring-shaped complexes with an external diameter of ∼9 nm and a central cavity of ∼2 nm (see magnifications 1–4 in Figure 7). Figure 7.Electron microscopy of C-IgAP. Electron micrographs (×60 000) of HEβ samples (0.5 mg/ml) stained with (A) 2% ammonium molybdate or (B) 2% uranyl acetate. The white bar corresponds to 35 nm. Images 1 and 2 were magnified from (A), and images 3 and 4 were magnified from (B). The black bar corresponds to 10 nm. Download figure Download PowerPoint The N-domains of a complex are secreted through a common channel The remarkable consistency between the size of the central pore of the C-IgAP complex observed by electron microscopy and that of the hydrophilic pore measured by liposome swelling suggested that this was the actual site used for the secretion of the N-domains in the complex. To investigate this, it was determined whether the simultaneous production of two distinct N-domains could interfere with their secretion by C-IgAP. The rationale behind this experiment was the assumption that if a common pore is used, a bulky N-domain blocking it would hinder the passage of a small N-domain that would otherwise be secreted efficiently. Thus, two hybrids of C-IgAP, with N-domains with different surface display properties, were co-expressed from compatible plasmids in the same E.coli cell. One of these hybrids was FvHβ, which appeared to block the hydrophilic channel of C-IgAP (see above). The other hybrid selected was Hβ, which completely translocates its N-domain (a His6 epitope) towards the surface of E.coli. Hβ is a derivative of HEβ in which the E-tag was deleted. In this way, FvHβ and Hβ hybrids could be detected independently after their co-expression in E.coli by employing different mAbs, anti-E-tag and anti-His, respectively. The expression and surface display of the two hybrids in E.coli UT5600 first was analyzed independently. Whole-cell ELISA with anti-His mAb (data not shown) and trypsin accessibility assays (Figure 8A, lanes 1–4) showed that ∼100% of the His6 epitopes were displayed on the surface of E.coli cells producing Hβ. This was clearly revealed by the full accessibility of the His6 peptide to externally added trypsin (compare lanes 1 and 3). In contrast, only 15–20% of the FvHβ produced in E.coli cells exposed the N-scFv on the surface, as judged by whole-cell ELISA with anti-E-tag mAb (Veiga et al., 1999) and trypsin digestion (Figure 8A, lanes 9–12). Most of the N-scFv (∼80%) was protected from proteolysis in intact cells (compare lanes 9 and 11). Importantly, trypsin was not able to degrade the N-scFv fully even after permeabilization of the OM with an EDTA shock (Figure 8A, lanes 10 and 12), indicating that this domain was probably embedded in the OM. Figure 8.Interference in the translocation of N-domains. (A) The C-IgAP hybrids Hβ and FvHβ were co-expressed (or expressed independently) in E.coli UT5600 cells. These cells were shocked with 10 mM EDTA or incubated with trypsin (1 μg/ml) as indicated (+). The digestion of Hβ and FvHβ was monitored by western blots using anti-His or anti- E-tag mAbs. The amount of OmpA (detected with rabbit anti-OmpA serum) was used as a loading control. (B) The subcellular location of Hβ and FvHβ in E.coli UT5600 cells producing Hβ alone or in combination with FvHβ is shown. The total protein extracts from these E.coli cells were separated into soluble (S), inner membrane (IM) and outer membrane (OM) protein fractions, as described previously (Veiga et al., 1999). OmpA was used as a control of the OM fractions. The proteins were detected by western blot as in (A). Download figure Download PowerPoint The surface display properties of the His6 epitope in Hβ changed dramatically when the two hybrids were co-expressed in E.coli UT5600 cells. In this case, the N-passenger of Hβ was almost completely protected (∼95%) from trypsin digestion in intact E.coli cells (Figure 8A, lanes 5 and 7), clearly supporting the hypothesis that the same pore was being used by the translocation of the N-scFv and the His6 N-passenger domains. Indeed, the His6 was partially protected from the protease even when the cells were shocked with EDTA (Figure 8A, lane 8). Three independent induction experiments with E.coli cells carrying either one or both plasmids were performed to confirm these results. OmpA was used as an internal control