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Keywords:

  • assembly;
  • outer membrane protein;
  • folding;
  • intermediates;
  • folded monomer

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse–chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, ΔF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the ΔF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic β strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.

Abbreviations
LPS

lipopolysaccharide

LDAO

N,N-dimethyldodecylamine-N-oxide

OMPs

outer membrane proteins

IPTG

isopropyl thio-β-d-galactoside

GdnHCl

guanidine hydrochloride

PtdCho

phosphatidylcholine

The cell envelope of Gram-negative bacteria consists of two membranes, the inner and the outer membrane, which are separated by the periplasm. To reach their destination, integral outer membrane proteins (OMPs) have to be transported across the inner membrane via the Sec machinery [1]. As hydrophobic sequences would act as stop-transfer signals [2,3], OMPs do not contain such sequences. Hence, the structure of OMPs is entirely different from those of integral inner membrane proteins, which contain hydrophobic α helices as the prominent membrane-spanning segments. Recently, the crystal structures of several OMPs have been solved [4–12]. All these proteins are β barrels, consisting of 8–22 antiparallel, amphipathic β strands that span the membrane, with the hydrophobic side exposed to the lipids.

Whereas the mechanism of the transport of proteins across the inner membrane via the Sec machinery has already been studied intensively in the past decades, the knowledge concerning the later steps in the biogenesis of OMPs is still very limited. After their translocation as linear polypeptide chains across the inner membrane, they probably fold in the periplasm prior to their insertion into the outer membrane [13]. Several periplasmic proteins, including SurA [14,15] and Skp [16–18] that may play a role in the folding of OMPs or in their targeting to the outer membrane have been identified, but their exact role is unknown. In addition, lipopolysaccharide (LPS), a major lipidic component of the outer membrane, has been implicated in the folding of OMPs [19–22]. It is unknown whether OMPs insert spontaneously into the outer membrane or whether a proteinaceous machinery is implicated in this process.

In our laboratory, PhoE protein of Escherichia coli is used as a model for studying the biogenesis of OMPs. The structure of this trimeric porin has been solved [5]. Previously, we have studied the assembly of this porin extensively after its synthesis in vitro in an S135 lysate of E. coli. In the presence of low amounts of Triton X-100 (0.015%, i.e. at the critical micelle concentration), PhoE could be assembled into a folded monomer that displays a higher electrophoretic mobility in SDS/PAGE than the denatured protein; LPS and divalent cations were required for the formation of this form of the protein [23]. Upon addition of outer membranes and increasing the detergent concentration to 0.08%, these folded monomers could be converted into stable trimers, which were incorporated in the membranes, suggesting that the folded monomer represents a bona fide assembly intermediate of the PhoE protein. On the basis of these in vitro experiments, it was suggested that the assembly of PhoE proceeds sequentially in the order: (a) formation of the folded monomer; (b) sorting to assembly sites in the outer membrane; (c) trimerization; and (d) insertion [23].

Especially in view of the requirement for detergent, it is not clear whether the results of the in vitro experiments indeed reflect the events in the PhoE assembly process in vivo. Therefore, any conclusion of these in vitro data has to be verified by in vivo experiments. The existence of a folded monomer of PhoE could be confirmed in vivo in pulse–chase experiments, followed by immunoprecipitation [24]. However, because of the slow kinetics with which these folded monomers disappeared during the chase, it was not immediately clear whether this form of the protein indeed represents an assembly intermediate.

In the present work, we have studied the assembly of PhoE in vivo more extensively. The goal was to detect assembly intermediates of the protein and to determine their subcellular localization. Furthermore, an in vitro system with purified components would be extremely beneficial to gain insight in the assembly process. Hence, in this paper, we describe an efficient procedure for the folding of PhoE after its production in inclusion bodies. In addition to the wild-type protein, the assembly of two mutant proteins was studied in vivo and in vitro.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Bacterial strains

The E. coli strain CE1224 is deleted for the phoE gene and does not express the related OmpF and OmpC proteins because of an ompR mutation [25]. The E. coli strain BL21(DE3) contains a copy of the T7 RNA polymerase gene under control of the lacUV5 promoter on the chromosome [26]. S135 cell extracts and outer membranes were isolated from the E. coli strain MC4100 [27].

Plasmids

The plasmids used in this study are listed in Table 1. Plasmid pJP29, containing the phoE gene, was digested with PstI (located at the border of the signal sequence and the mature protein-encoding DNA) and treated with T4 DNA polymerase and dNTPs to create blunt ends. Subsequently, the plasmid was digested with SalI, and a 10-mer linker containing an NcoI restriction site was cloned upstream of the DNA encoding the mature part of PhoE, resulting in plasmid pCJ1. Plasmid pCJ1 was digested with NcoI and ClaI (located in phoE), and the resulting fragment was substituted for the NcoI–ClaI fragment in pT7phoE, resulting in plasmid pCJ2. Plasmid pT7phoE consists of the BamHI–PvuI fragment of pNN100 cloned into pET11d in the corresponding restriction sites. The mutations ΔF330 and G185E were introduced in pCJ2 by substitution of NsiI fragments of pMS26 and pMS01, respectively, for the corresponding fragment of pCJ2, resulting in plasmids pCJ7 and pCJ15, respectively.

Table 1. Plasmids and their relevant characteristics and sources.
PlasmidRelevant characteristicsReference/source
pET11dExpression vector, T7 promoter[28]
pJP29Wild-type phoE gene behind the phoE promoter[29]
pCJ1Derivative of pJP29 with a linker substituted for the promoter and signal sequence-encoding part of phoEThis study
pT7phoEWild-type phoE gene behind T7 promoter of pET11dN. Nouwen
pNN100Wild-type phoE behind tac promoter of pJF118EH[30]
pCJ2Wild-type phoE without the signal sequence-encoding segment behind T7 promoter of pET11dThis study
pMS26pJP29 derivative with a mutation resulting in the deletion of C-terminal Phe of PhoE (ΔF330)[31]
pMS01pJP29 derivative with a mutation resulting in a G185E subsitution in PhoE[32]
pCJ7Mutant phoE gene (ΔF330) without the signal sequence-encoding segment behind T7 promoter of pCJ2This study
pCJ15Mutant phoE gene (G185E) without the signal sequence-encoding segment behind T7 promoter of pCJ2This study
pNN100ΔF330Mutant phoE gene (ΔF330) behind tac promoterThis study
pNN100G185EMutant phoE gene (G185E) behind tac promoterThis study

Plasmid pNN100 contains the phoE gene under control of the tac promoter. The mutant phoE plasmids pNN100G185E and pNN100ΔF330 were constructed by substituting a PstI–BglII fragment from plasmid pMS01 and a BglII–PvuI fragment from pCJ7, respectively, for the corresponding fragments of pNN100. Mutations were confirmed by double-stranded DNA sequencing using the ABI Prism™ 310 Genetic Analyzer (PerkinElmer).

Expression and in vitro folding of PhoE

Cultures of strain BL21(DE3) containing appropriate plasmids, grown overnight at 37 °C in Luria–Bertani medium supplemented with ampicillin (50 µg·mL−1), were diluted 1 : 10 into fresh Luria–Bertani medium supplemented with ampicillin and 0.5% glucose. When the D660 reached a value of 0.6, isopropyl thio-β-d-galactoside (IPTG) was added to a final concentration of 1 mm. After 3 h further incubation at 37 °C, the cells were harvested, resuspended in 10 mm Tris/HCl, pH 8.0, 3 mm EDTA and disrupted by sonication (2 × 5 min at level 7, 40% output, Branson sonifier 450) and the inclusion bodies were collected by centrifugation (15 min, 2400 g, SS34-rotor) and dissolved in 6 m guanidine hydrochloride (GdnHCl). Ultracentrifugation (2.5 h, 145 000 g, Ti-50 rotor) was used to remove residual membrane fragments. The supernatant was stored at 4 °C.

Folding of PhoE was achieved by diluting the GdnHCl-denatured inclusion bodies in 20 mm Tris/HCl, pH 7.0, 0.2% (w/v) N,N-dimethyldodecylamine-N-oxide (LDAO, Fluka) (final concentrations: 100 µg·mL−1 protein, 0.12 m GdnHCl). The mixture was incubated overnight at room temperature (or at 0 °C). Prior to analysis by SDS/PAGE and Western blotting, the residual amounts of GdnHCl were removed by dialysing the PhoE folding mixture for 3 h at room temperature (or at 4 °C when folding was performed at 0 °C) against 10 mm Tris/HCl, pH 8.0, 0.2% LDAO.

Pulse–chase experiments

Cultures of strain CE1224 containing pNN100 or derivative plasmids were grown at 37 °C in minimal medium [33] with 0.5% glucose as a carbon source and supplemented with requirements for growth due to auxotrophic mutations at appropriate concentrations and 50 µg·mL−1 ampicillin. After overnight growth, the cultures were diluted 10-fold in the same medium and grown for 4 h at 30 °C. PhoE expression was induced by the addition of IPTG (0.1 mm final concentration). After 30 min incubation at 30 °C, the cells were pulse-labeled with [35S]-Pro-mix (Amersham Pharmacia Biotech) for 30 s (85 µCi per 8 mL of cells), followed by a chase with an excess of nonradioactive methionine/cysteine. After various time intervals, 1.5-mL samples were transferred into microtubes precooled on ice. During the following procedures, samples were kept ≤ 4 °C. Labeled cells were pelleted by centrifugation and resuspended in 0.5 mL of 10 mm Tris/HCl, pH 8.0. Cell envelopes were isolated by ultracentrifugation (30 min, 70 000 g, TLA 100.2 rotor, Beckman) after ultrasonic disintegration of the cells (3 × 10 s at 60 W; Branson sonifier) and resuspended in 100 µL of 10 mm Tris/HCl, pH 8.0. Supernatants contained the soluble cytosolic and periplasmic proteins.

Folding and assembly of in vitro synthesized PhoE protein

Isolation of S135 cell extracts and the in vitro transcription and translation reactions were performed as described [34] with the following modifications. The plasmids from the pCJ series were transcribed with T7 RNA polymerase (70 U·µL−1, Pharmacia) in a transcription mix containing 20 mm Hepes, pH 7.5, 10 mm MgAc, 2 mm spermidine, 0.5 mm NTPs, RNAse inhibitor (24 U per 100 µL), 25 mm NaCl and 5 mm dithiothreitol. Incubations were carried out for 1 h at 37 °C. The translation assay contained 20 mm, instead of 60 mm potassium acetate. Folding of in vitro synthesized PhoE with purified LPS and 0.015% Triton X-100 and the subsequent assembly of folded monomers into outer membranes were performed as described [23]. Routinely, folded monomers were not treated with trypsin before assembly into the outer membranes. Samples were analysed by SDS/PAGE and autoradiography.

Trypsin digestion

Trypsin treatment was performed with 50 µg·mL−1 TosPheCH2Cl-treated trypsin (Serva) for 30 min at 37 °C (after in vitro folding) or at 0 °C (in the case of radiolabeled samples or after in vitro folding at 0 °C). The reaction was terminated by adding 1 mm phenylmethanesulfonyl fluoride and incubation on ice.

SDS/PAGE and Western blotting

Prior to electrophoresis, samples were incubated for 10 min at either room temperature, 56 °C or 100 °C in sample buffer [35] containing either 0.05% or 2% SDS and supplemented with 10 mm EDTA. SDS-polyacrylamide gels were prepared as described previously [35], except that the stacking and running gels contained no SDS. The gels were run at 20 mA in a temperature-controlled room at 4 °C to prevent denaturation of various folded forms of the PhoE protein during electrophoresis.

After SDS/PAGE, the gels loaded with radiolabeled samples were dried, treated in Amplify (Amersham) and exposed to X-ray films at −80 °C. Quantifications were performed using the program imagequant after exposing the gels in a Phosphor Imager (Molecular Dynamics). When the intensity of the PhoE band at the position of the denatured monomer after incubating the sample at room temperature is designated ‘a’, at 56 °C ‘b’ and at 100 °C ‘c’, the proportion of stable trimers can be defined as (c − b)/c, the metastable forms as (b − a)/c and the unfolded monomer as a/c.

For analysis of the in vitro folding assay, Western blotting was performed as described previously [36]. Prior to the blotting procedure, gels were heated under steam [37] to denature folded proteins. The polyclonal antiserum used to detect PhoE, α-PhoE 882, was raised against the denatured protein.

Urea extraction

Cell envelopes (25 µL) were mixed with 75 µL of 8 m urea in 10 mm Tris/HCl, 50 mm glycine, pH 8.0, and incubated on ice for 30 min. Cell envelopes were re-isolated by ultracentrifugation (30 min, 70 000 g, TLA 100.2 rotor) at 4 °C and dissolved in 50 mm triethanolamine acetate, pH 7.5, 250 mm sucrose, 1 mm dithiothreitol.

Gradient centrifugations

Flotation gradient centrifugations in metrizamide (ICN) were performed as described [38] using gradients of 500 µL and centrifugation for exactly 5 h at 4 °C at 436 000 g in a TLA 100 rotor.

Sedimentation gradient centrifugation was performed as described [38], with the modification that the sucrose solutions were buffered with 50 mm triethanolamine acetate, pH 7.5, and contained 1 mm EDTA and 1 mm dithiothreitol. Proteins from the fractions obtained were concentrated by precipitation with 5% trichloroacetic acid and washed with acetone. The pellet was dried, resuspended in sample buffer and analysed by SDS/PAGE.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Assembly of wild-type PhoE protein in vivo

Pulse–chase experiments were performed to study the kinetics of the assembly of the PhoE protein and to identify putative assembly intermediates. Instead of 37 °C, we used 30 °C as the growth temperature in order to slow down the assembly process. After the pulse and the chase, the samples were rapidly cooled at 0 °C to stop the maturation of the PhoE protein. To determine the subcellular location of the different forms of PhoE detected, the membranes were separated from the soluble fraction by centrifugation after disruption of the cells. The cell envelopes (pellet) and the soluble (supernatant) fractions were analysed by SDS/PAGE either directly or after trypsin digestion. In contrast to the common practice in almost all other OMP assembly studies, immunoprecipitations were not used in the present study to avoid the possibility that certain folding intermediates would be missed because of nonrecognition by the antibodies used. Furthermore, it was shown that in vitro synthesized PhoE is able to fold during the immunoprecipitation procedure due to the presence of detergents that are included in the immunoprecipitation buffers to avoid unspecific aggregation [39]. A second reason to omit the immunoprecipitations is that conversion of assembly intermediates to other folded states is not desirable. We revealed the identity of the PhoE protein bands by comparison of the SDS/PAGE protein patterns with those of a strain not expressing the PhoE protein.

The total amount of radiolabeled PhoE protein increased during the chase (Fig. 1A). This is probably (partly) due to the completion of nascent chains of PhoE, resulting from translation initiated within the 30-s pulse period. The majority of the PhoE molecules present directly after the pulse was detected in the pellet fraction after ultracentrifugation of disintegrated cells (Fig. 1A). Moreover, more than half of the PhoE molecules in this fraction were already assembled in the stable trimeric configuration, which withstands heating in 2% SDS at 56 °C (Fig. 2A,B) and protease-treatment (Fig. 2C,E). This form was integrated in the membrane, as it could not be extracted with urea (results not shown). The total amount of the stable trimeric form further increased during the chase (Fig. 2B,E). A significant amount of the PhoE protein was assembled into a metastable folded form, which denatured by heating at 56 °C (Fig. 2A) and includes mainly folded monomers (designated FM, which migrate with an apparent molecular mass of 31 kDa) and possibly also labile trimers that dissociate at a lower temperature than the mature trimers. In addition, these metastable forms of the protein appeared to be resistant to trypsin (Fig. 2C) and to urea extraction (results not shown). Their amounts decreased during the chase with rather slow kinetics (half-time of several min) (Fig. 2B,E), suggesting that they were converted into stable trimers. Finally, a small amount of a PhoE-specific band was detected directly after the pulse in the position of the non-native monomeric form, i.e. with an apparent molecular mass of 37 kDa (Fig. 2A, lanes a). This non-native form was digested by trypsin (Fig. 2C), indicating that it had not obtained a compact folded conformation. This form could be extracted from the membranes with urea (results not shown), demonstrating that it was not integrated into, but peripherally associated with, the membranes. Its amount gradually decreased during the chase (Fig. 2B), indicating that it was converted to the stable PhoE forms, but the alternative possibility, that it is proteolytically degraded during the chase, cannot be excluded at this stage. As expected, sedimentation gradient centrifugation revealed that the vast majority of PhoE proteins in the pellet fraction was present in the outer membranes rather than in the inner membranes (results not shown).

image

Figure 1. Relative amounts of radioactively labeled wild-type (A) and ΔF330 (B) PhoE in the soluble and membrane fractions during pulse–chase experiments. After pulse-labelling of the cells and chase for various time intervals, the highest amount of total radioactively labeled PhoE detected was arbitrarily chosen to be 100% and the amounts of PhoE protein at the remaining time points (and in the different fractions) were related to this amount. The values are obtained from one representative experiment.

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image

Figure 2. Pulse–chase experiment with cells expressing wild-type (A-E) and G185E (F) mutant PhoE: analysis of the pellet fraction.E. coli CE1224 cells containing pNN100 or pNN100G185 were pulse-labeled and chased with cold methionine/cysteine. Cell envelopes were isolated and either directly analysed by SDS/PAGE (A), or first subjected to trypsin treatment (C,D,F). Samples were analysed at the indicated time points post pulse. Each sample was divided into three equal aliquots and incubated for 10 min at room temperature (a), 56 °C (b) or 100 °C (c) in the presence of 2% SDS (A,C,F) or 0.05% SDS (D) prior to electrophoresis. The positions of the trimers (T), non-native monomers (M) and folded monomers (FM) are indicated. Positions of molecular mass standard proteins are indicated at the left of (A) (in kDa). (B) and (E) show the quantification of experiments shown in (A) and (C), respectively. The values are the mean of 12 independent experiments.

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The folded monomeric form detected in the experiments described above might exist in vivo as a true monomeric assembly intermediate. Alternatively, it might have emerged from unstable trimers, which dissociated upon sample preparation. To investigate these possibilities, we also incubated the samples with lower amounts of SDS before SDS/PAGE. When the trypsin-treated samples were incubated in sample buffer with 0.05% instead of 2% SDS, the folded monomers were detected at low levels if at all (Fig. 2D, only shown for t = 10 s), indicating that this form emerged from SDS-sensitive trimers at high SDS concentrations.

Directly after the pulse, a significant amount of PhoE was detected in the supernatant fraction after ultracentrifugation of the disintegrated cells, and this amount slightly decreased during the chase (Fig. 1A). Analysis of this fraction by SDS/PAGE revealed the presence of a non-native PhoE form directly after the pulse (Fig. 3B, lane a), which was sensitive to trypsin (Fig. 3D, lane a). The amount of this form was decreased after 15 min chase (Fig. 3C, lane a). In addition, a small amount of trypsin-resistant folded forms of PhoE was detected in the soluble fraction directly after the pulse (Fig. 3D), which also slightly decreased during the chase (Fig. 3E). These trypsin-resistant PhoE forms may be present in small membrane vesicles that were not pelleted during the ultracentrifugation. Both flotation and sedimentation gradient centrifugation indeed revealed that part of the PhoE protein in the supernatant fraction was associated with membranes (results not shown). The non-native and trypsin-sensitive form probably reflects a soluble unfolded monomeric intermediate.

image

Figure 3. Pulse–chase experiment with cells expressing wild-type PhoE: analysis of the supernatant fraction.E. coli CE1224 cells without (A) or with (B-E) pNN100 were pulse-labeled and chased with cold methionine/cysteine. The supernatant, obtained after ultracentrifugation of disintegrated cells, was either directly analysed by SDS/PAGE (A–C) or after trypsin-treatment (D–E). The samples were incubated at room temperature (a), 56 °C (b) or 100 °C (c) in the presence of 2% SDS. The position of the monomeric PhoE band is indicated by an arrow (B–E). In (A), the arrow indicates the corresponding position. Positions of molecular mass standard proteins are indicated at the left (in kDa).

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In conclusion, about half of the total PhoE molecules assembled with very fast kinetics in the membrane, while the remaining molecules showed much slower kinetics. Among the latter molecules, three putative assembly intermediates were detected: (a) a soluble non-native form; (b) a membrane-associated non-native form; and (c) a metastable folded form, probably representing labile trimers, rather than folded monomers.

In vitro folding of the wild-type PhoE protein

Initially, we attempted to fold the PhoE protein isolated from inclusion bodies according to the procedures developed previously for the porin PorA of Neisseria meningitidis[40]; this was not sucessful (results not shown). Hence, other folding strategies had to be followed. We applied the conditions described by Eisele and Rosenbusch [41] for the rapid refolding of the closely related porin OmpF, purified from the outer membrane of E. coli. According to this procedure, folding is accomplished in mixed micelles containing phosphatidylcholine (PtdCho) and octyl-poly-oxyethylene. Although folding of PhoE was achieved (results not shown), the folding efficiencies were very poorly reproducible and ranged from almost 0 to 90%. Because we intended to study the folding efficiencies of mutant forms of PhoE in vitro, the variable results obtained for the wild-type protein rendered this method unsuitable. Another strategy was based on the observation that in vitro synthesized PhoE was able to trimerize in a cell lysate upon addition of detergents containing a phenyl ring [39]. In accordance with these findings, the PhoE protein, isolated from inclusion bodies, folded into a trimeric form in the presence of 2% Triton X-100; however, the efficiency was only 5% (results not shown).

Finally, we succeeded in folding the PhoE protein efficiently and reproducibly in the presence of the detergent LDAO. The folding of PhoE was analysed by SDS/PAGE and Western blotting. After in vitro folding of wild-type PhoE, heat-stable trimers were detected (Fig. 4, lanes 1 and 2). Furthermore, significant amounts of folded monomers and small amounts of dimers were observed (lane 1), both of which denatured at 56 °C (lane 2). Incubation of the sample in 0.05% SDS instead of 2% SDS still showed the dimeric and folded monomeric protein bands next to the trimeric forms (not shown), indicating that these forms did not emerge from labile trimers. Next to these folded forms, a small amount of non-native PhoE was detected (Fig. 4, lane 1) which was trypsin-sensitive (results not shown). However, the vast majority of the in vitro folded PhoE was trypsin-resistant (Fig. 4, lane 4), indicating that the protein is correctly folded [42]. Remarkably, folding at 0 °C (and subsequent dialysis at 4 °C) also resulted in the efficient formation of folded, trypsin-resistant protein (not shown), indicating that the folding in LDAO is driven by enthalpy rather than entropy.

image

Figure 4. In vitro folding of guanidine-denatured PhoE, wild-type (WT) andΔF330. Folding occurred in 0.2% LDAO (see Materials and methods). Samples were trypsin treated (+) or not (–) and incubated at room temperature (a), 56 °C (b) or 100 °C (c) prior to SDS/PAGE. The positions of trimers, dimers, monomers and folded monomers (monomer*) are indicated at the left. Positions of molecular mass standard proteins are indicated at the right (in kDa).

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Folding and assembly of the ΔF330 mutant PhoE protein

The vast majority of bacterial OMPs contain a Phe (or Trp) at the C-terminal position, indicating an important role for this amino acid in outer membrane assembly. Indeed, the deletion of the C-terminal Phe of PhoE resulted in a severe assembly defect in vivo[31]. To determine the exact step that is defective in the assembly of this mutant protein, we performed pulse–chase experiments and separated soluble and membrane proteins by ultracentrifugation after disruption of the cells. In contrast to the results described for the wild-type protein, the majority of the radioactively labeled ΔF330 proteins detected directly after the pulse was present in the supernatant fraction (Fig. 1B). No significant amounts of folded, trypsin-resistant forms were detected in this fraction, and sedimentation gradient centrifugation revealed that hardly any of this protein was associated with lipids (results not shown). This soluble intermediate was partly chased into the pellet fraction during 15 min chase (Fig. 1B). Apparently, the deletion of the C-terminal Phe resulted in the accumulation of large amounts of a soluble unfolded intermediate, which was slowly chased into the membrane fraction.

The majority of PhoE in the pellet fraction was non-native (Fig. 5A, lane a), trypsin-sensitive (Fig. 5B, lane a) and extractable from the membrane with urea (results not shown). Only low amounts of folded, trypsin-resistant proteins were detected directly after the pulse, but this amount increased steadily during the chase (Fig. 5B). No folded monomeric form with an apparent molecular mass of around 31 kDa was detected. Previously, it has been demonstrated that the folded monomers of ΔF330 were less stable than those of the wild-type protein, and that they denatured during SDS/PAGE, resulting in a smear of protein migrating between 31 and 37 kDa [43]. Hence, we assume that the folded monomers of the ΔF330 PhoE protein might also have been present in our pulse–chase experiments, but could not clearly be detected on SDS/PAGE. In conclusion, the deletion of the C-terminal Phe directed the PhoE protein in vivo largely to the slower kinetic assembly pathway, comprising an unfolded soluble intermediate.

image

Figure 5. Pulse–chase experiment with cells expressingΔF330 mutant PhoE: analysis of the pellet fraction.E. coli CE1224 cells containing pNN100ΔF330 were pulse-labeled and chased with cold methionine/cysteine. Cell envelopes were isolated and subjected to trypsin treatment (B) or not (A) and analysed by SDS/PAGE. Samples were incubated in sample buffer containing 2% SDS at room temperature (a), 56 °C (b) or 100 °C (c) prior to SDS/PAGE. The positions of the trimers (T) and non-native monomers (M) are indicated. Positions of molecular mass standard proteins are indicated at the left of (A) (in kDa).

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After isolation from inclusion bodies, the ΔF330 mutant PhoE protein folded in the presence of LDAO mainly into trimers, which were resistant to heating at 56 °C (Fig. 4, lanes 5 and 6). Folded monomers were not detected as a defined band on SDS/PAGE, but a smear of protein was observed between the positions of the folded monomer and the denatured monomer, probably as a result of denaturation of folded monomers during electrophoresis and reflecting the reduced stability of this form of the mutant protein. Virtually all ΔF330 protein was resistant to trypsin after folding, indicating that the protein had acquired the correct conformation. In addition, the ΔF330 mutant protein was hardly affected in folding in this in vitro system at 37 °C (not shown).

Folding and assembly of the G185E mutant protein

To determine whether hydrophilic residues are tolerated at the protein–lipid interface, we introduced a charged residue, glutamic acid, at the hydrophobic side of the β strand 9 resulting in the mutant protein G185E. When analysed in vivo in pulse–chase experiments, large amounts of the G185E mutant protein appeared as trypsin-resistant trimers in the membrane fraction directly after the pulse (Fig. 2F). This amount increased further during the chase (results not shown). Thus, even though the substitution G185E is a very drastic one, introducing a charged residue at the hydrophobic side of β strand 9, both the fast and the slow kinetic assembly pathways appeared to be operative for this mutant protein.

After its expression in inclusion bodies and subsequent solubilization in GdnHCl, the mutant protein G185E could be folded in LDAO into trimers, with an efficiency similar to the wild-type protein (results not shown). We also analysed the assembly of this mutant protein in a previously established in vitro assay [23]. In this assay, the protein is synthesized in a radioactive form in a cell-lysate and folded into folded monomers in the presence of 0.015% Triton X-100, LPS and divalent cations. Upon addition of outer membranes and increasing the detergent concentration to 0.08%, these folded monomers could be assembled into stable trimers, which were integrated in the membranes. Remarkably, whereas the wild-type protein was able to form trypsin-resistant folded monomers with LPS and Triton, the G185E mutant protein was severely defective in this step (Fig. 6). However, those folded monomers that were formed could subsequently be converted into trimers (Fig. 6).

image

Figure 6. Folding and assembly of in vitro synthesized PhoE.In vitro synthesized PhoE was incubated with LPS and Triton X-100 (0.015%) to form folded monomers (each first panel). Assembly of trimers (each third panel) was induced by raising the Triton X-100 concentration to 0.08% and by the addition of isolated outer membranes. Trypsin treatment was performed after the formation of FMs, or, when assembly was performed, after the insertion into outer membranes. Prior to SDS/PAGE, samples were incubated for 10 min at room temperature (a), 56 °C (b) or 100 °C (c). The positions of trimers (T), non-native monomers (M) and folded monomers (FM) are indicated. The third panel of mutant G185E is exposed three times longer.

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In conclusion, while the mutation G185E was well tolerated in vivo and in the folding of the isolated protein in vitro with LDAO, the mutant protein was not able to form folded monomers with LPS and Triton after its synthesis in vitro.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

The assembly of outer membrane proteins in E. coli is a multistep process. We investigated the assembly of wild-type PhoE porin and of two mutant PhoE proteins in vivo and in vitro, in order to dissect the assembly process in separate steps.

Two pathways for the assembly process in vivo were revealed with the wild-type protein. Approximately 50% of the wild-type PhoE molecules synthesized followed very fast assembly kinetics. Directly after pulse-labelling of the cells, these molecules were recovered in the membrane fraction in a stable trimeric and trypsin-resistant conformation, which can be considered as the native conformation of the protein. The other half of the wild-type PhoE molecules followed a slower kinetic pathway. A possible explanation for the detection of two kinetically different assembly pathways is that the PhoE protein was overproduced and, as a consequence, was titrating out a putative chaperone. Consistent with this hypothesis is the observation that the proportion of the PhoE molecules that followed the fast kinetics increased when the synthesis level was reduced (data not shown). Because the fast kinetic assembly pathway appeared to be severely disturbed in the ΔF330 mutant protein, we propose that the C-terminal Phe, which is conserved among most bacterial OMPs, is (part of) the targeting signal that is recognized by the putative chaperone. This targeting signal is not necessary for progress in the slow kinetic assembly pathway. The folding of the ΔF330 mutant protein was not affected in the established in vitro system with LDAO, indicating that the protein is intrinsically able to adopt a native-like structure. This observation underscores the hypothesis that the ΔF330 mutant protein is defective in the fast kinetic assembly pathway because of the deletion of (part of) a targeting signal rather than being disturbed in folding itself. The TolA and TolB proteins have been reported to interact with folded porin molecules [44,45]. Hence, these Tol proteins might be the chaperones that target PhoE to the outer membrane.

Three different intermediates could be distinguished in the slow kinetic assembly pathway. Small amounts of a soluble form were detected. This form is probably located in the periplasm, as the signal sequence was cleaved off. The observation that the periplasmic DsbA protein could introduce disulfide bonds in a cystine-containing mutant form of PhoE [13] is consistent with the idea that PhoE passes through the periplasm on its way to the outer membrane. Furthermore, as the DsbA protein creates an element of tertiary structure, the mutant PhoE was at least partially folded prior to insertion into the outer membrane [13]. Consistently, we detected unfolded as well as folded forms in the periplasm. However, a proportion of the proteins detected in the supernatant fraction appeared to be associated with lipids. Because of the small quantities of these proteins, we were not able to establish definitely whether the lipid-associated PhoE molecules corresponded to the trypsin-resistant folded forms, but this seems a plausible assumption. A water-soluble monomeric intermediate has also been reported for the OmpF protein [46]. This form was secreted by spheroplasts and could be converted to trimers upon the addition of cell envelopes and small amounts of detergent, indicating that it was a real assembly intermediate. Furthermore, the periplasmic SurA protein has been observed to be involved in the conversion of unfolded monomers of LamB into folded monomers [15], consistent with the idea that folding is a periplasmic or periplasmically exposed membrane-associated event.

A second assembly intermediate detected was a non-native, trypsin-sensitive form, which was loosely associated with the membranes. As the proteins in the fractions obtained after sucrose gradient centrifugation had to be recovered by trichloroacteic acid precipitation, which denatures all folded forms, we were not able to identify whether this non-native form was associated with the inner or outer membrane. It is possible that the non-native intermediate is associated with the inner membrane and precedes the soluble intermediate. In accordance with this supposition is the observation that processed intermediates in the assembly of LamB have been localized in the inner membrane [47]. Furthermore, an immature processed form of OmpA that accumulated after overproduction was probably associated with the inner membrane [48].

The third assembly intermediate detected in vivo was a trypsin-resistant metastable trimer, which dissociated in folded monomers in 2% SDS. This form was integrated into the membranes, as it could not be extracted with urea, and it was converted to the stable trimeric configuration with relatively slow kinetics. Previously, metastable trimers of LamB have been detected, which were stabilized with a half-time of several min [49]. Also in the case of OmpF, the existence of a metastable trimeric intermediate, localized in the periplasm, has been reported [50]. However, as this form was detected in the periplasmic fraction by immunoprecipitation in the presence of 1% Triton X-100, the possibility that this form was only formed in vitro during the immunoprecipitation procedure from an unfolded intermediate can not be excluded. As suggested for the LamB protein [49], the stabilization of the trimers might be induced by the increased interaction with other outer membrane components, such as LPS. Consistently, the stabilization of OmpF and LamB trimers appeared to be affected in deep-rough mutants of E. coli[22].

Importantly, although we did detect folded monomers on gels, most, if not all, appeared to be generated by the dissociation of metastable trimers. Probably, the folded monomers of PhoE and LamB that were previously detected in vivo[15,24,51] were derived from SDS-sensitive trimers as well. In contrast, the folded monomers that are formed in vitro by incubation of de novo synthesized protein with LPS and a low concentration of Triton X-100 [23] represent a real monomeric form, as incubation in 0.05% instead of 2% SDS prior to electrophoresis did not reveal any oligomeric bands on SDS/PAGE (H. de Cock, unpublished observation). Moreover, because this in vitro formed folded monomer could subsequently be trimerized upon the addition of outer membranes, it is an assembly intermediate, at least in vitro. However, the failure to detect a folded monomer in vivo in the pulse–chase experiments could mean that such a form of PhoE does not exist as a bona fide assembly intermediate in vivo, and that folding of the porins is initiated by the assembly of the trimeric subunit interface, as has been suggested previously [52]. Consistent with this hypothesis are the results obtained with the G185E mutant. The introduction of a charged residue at the hydrophobic side of β strand 9 appeared to be tolerated in vivo and during folding in vitro in LDAO. However, the G185E mutant protein failed to form folded monomers with LPS and Triton after its synthesis in vitro. These results again strongly suggest that the folded monomer is not a true folding intermediate in vivo.

We therefore propose the following model for the assembly pathway of PhoE. After translocation across the inner membrane, the signal sequence is cleaved off, producing a mature PhoE protein, which is very rapidly converted to the native trimeric conformation in the outer membrane, probably in a chaperone-mediated process. The ΔF330 mutant protein is blocked in this process. This mutant protein and, probably as a consequence of overproduction, a proportion of the wild-type molecules, follow slower kinetics. We suppose that the different stages detected in the slower kinetic pathway still reflect the stages in the fast kinetic pathway, but that the process is retarded due to limited interactions with one or more chaperones or with assembly sites in the outer membrane. Three intermediates in this slower pathway were detected in the pulse–chase experiments. We propose that the membrane-associated, non-native monomer is still associated with the inner membrane, from where it is released to create the soluble non-native monomer. Subsequently, the periplasmic intermediate is sorted to the outer membrane, where it is converted to a metastable trimer, which inserts into the membrane and is converted within minutes to the stable trimeric configuration. Further analysis of an array of PhoE mutants that is available in our laboratory in the developed in vivo and in vitro assembly assays will be helpful to verify the proposed working model.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References