Signal Sequence Mutations as Tools for the Characterization of LamB Folding Intermediates

Abstract

lamBA23DA25Y and lamBA23YA25Y tether LamB to the inner membrane by blocking signal sequence processing. We isolated suppressors of lamBA23DA25Y and lamBA23YA25Y, all of which mapped within the LamB signal sequence. Most interesting were mutations that changed an amino acid with a strong positive charge to an amino acid with no charge. Further characterization of two such suppressors revealed that they produce functional LamB that is localized to the outer membrane with its entire signal sequence still attached. Biochemical analysis shows that mutant LamB monomer chases into an oligomeric species with properties different from those of wild-type LamB trimer. Because assembly of mutant LamB is slowed, these mutations provide useful tools for the characterization of LamB folding intermediates.

In Escherichia coli, extracytoplasmic proteins must be targeted to three distinct compartments—the periplasm and the inner and outer membranes. All proteins in E. coli are initially synthesized in the cytoplasm, but the path that they then follow depends upon their ultimate cellular destination. Many proteins bound for extracytoplasmic locations are synthesized with an N-terminal signal sequence that directs them to the general secretion machinery at the inner membrane (for a review, see reference 12).

Two models dominate studies of outer membrane protein targeting. The first of these is the Bayer's junction model, which proposes that proteins travel to the outer membrane through zones of adhesion between the inner and outer membranes (1). The second model, known as the periplasmic intermediate model, predicts that outer membrane proteins pass through the secretion machinery at the inner membrane and into the periplasm before becoming localized to the outer membrane (31). The periplasmic intermediate model might suggest that proteins transiting through the periplasm are met by factors that aid in their folding. Indeed, several different groups of periplasmic folding factors have been identified. They include proteins involved in the formation and isomerization of disulfide bonds, peptidyl-prolyl cis-trans isomerases, and chaperones (2, 3, 7, 12, 13, 21, 27, 29, 32, 34, 36, 40).

Generally speaking, there are three major types of proteins in the outer membrane: surface organelles such as pili, lipoproteins, and the β-barrel proteins. The targeting of many pili is known to involve the periplasmic chaperone/usher pathway (37). Recently, the targeting of outer membrane lipoproteins has been elucidated (24, 25). Lipoproteins are complexed with a periplasmic protein, LolA, which carries them to the outer membrane, where they are met by the outer membrane-associated component, LolB. LolB is intimately involved with the incorporation of lipoprotein into the outer membrane (24, 25).

The folding and targeting of the β-barrel proteins is less well understood. These proteins include the outer membrane porins PhoE, OmpF, OmpC, and LamB. Our model for studying β-barrel proteins is LamB, the porin utilized for the import of maltodextrins and the cell's receptor for bacteriophage λ. The structure of the LamB porin has been elucidated, and it demonstrates that LamB forms an 18-stranded β-barrel in which each strand is amphipathic with alternating hydrophobic and hydrophilic amino acid residues (38).

As with other extracytoplasmic proteins, the β-barrel proteins are initially synthesized in the cytoplasm with an N-terminal signal sequence (15). The LamB signal sequence consists of three major regions (42). At the N terminus, there are two basic amino acids. Following these positively charged residues is a stretch of hydrophobic amino acids that assume an α-helical structure when the signal sequence is looped into the inner membrane (16). At the C-terminal end of the signal sequence are one or more helix-breaking residues and a consensus site for cleavage by signal peptidase. The consensus cleavage site generally consists of any amino acid bound on both sides by alanine (A-X-A) (42). Although variations in the signal sequence are tolerable, replacement of either of these two alanines by a bulkier amino acid results in loss of cleavage by signal peptidase (10, 17). If processing of the signal sequence is blocked, the protein remains tethered to the inner membrane (9, 10, 17).

After signal sequence cleavage, β-barrel proteins, such as those mentioned above, are targeted very efficiently to the outer membrane (for a review, see references 12 and 31). Although β-barrel proteins exist in the outer membrane as trimers, several folding intermediates have been described. These assembly intermediates include the mature unfolded monomer, folded monomer, dimer, and metastable trimer (for a review, see reference 12). β-barrel dimers have been reported for OmpF and OmpC (33, 35). With respect to LamB, evidence for the existence of an unfolded monomer, metastable trimer, and mature trimer is clear (26, 33). Folded monomer intermediates of LamB have also been found in certain strain backgrounds (36), but the cellular location of this intermediate has remained a mystery.

To understand how outer membrane proteins are targeted and inserted in the outer membrane, mutations that slow down or prevent processing and targeting of LamB have been constructed (6, 10). In particular, lamBA23DA25Y and lamBA23YA25Y both contain two mutations that alter the signal sequence consensus cleavage site, preventing cleavage by signal peptidase. Thus, LamB effectively becomes tethered to the inner membrane (8). The consequences of this tethering are threefold. First, tethered LamB is unable to function as a pore for the import of maltodextrins or as a λ receptor. Cells carrying these mutations are Dex⁻ and λ^R. Tethering also prevents LamB folding, and as a consequence, it is degraded in the periplasm. Finally, tethered LamB is toxic to the cell, as evidenced by sensitivity to inducers such as maltose (10). The reasons for this toxicity are unknown.

In this study we report the isolation and characterization of functional suppressors of lamBA23DA25Y and lamBA23YA25Y. Using these mutants, we provide evidence that LamB oligomers are functional in the outer membrane even with the entire signal sequence still attached. Furthermore, we show that the mutations slow LamB assembly in the outer membrane significantly. This crippling of LamB assembly has enabled us to localize a key assembly intermediate in the cell.

MATERIALS AND METHODS

Media and reagents.

Media were prepared as described by Silhavy et al. (39) with the following exceptions. M63 liquid minimal medium was supplemented with 0.4% (wt/vol) sugars and 0.5% (vol/vol) Luria-Bertani (LB) broth. [³⁵S]methionine was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, Calif.). LamB and maltose-binding protein (MBP) antisera are from our laboratory stock (26). Formalin-fixed Staphylococcus aureus used for immunoprecipitations was purchased from CalBiochem. Enhanced chemiluminescence (ECL) Western blotting reagents were purchased from Amersham Life Science (Piscataway, N.J.).

Bacterial strains and microbiological techniques.

The bacterial strains used in this study are derivatives of E. coli K-12 strain MC4100 [F⁻araD139 Δ(argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR] (39). Standard microbiological methods used for P1 transduction have been described previously (39).

Western analysis.

For standard Western blot analysis, cells were grown overnight in glycerol minimal medium at 37°C. Cells were subcultured into fresh glycerol minimal medium and grown to early log phase, at which point LamB synthesis was induced with 0.4% (wt/vol) maltose for 1 to 2 h at 37°C. Alternatively, cells were grown to saturation in LB liquid medium and subcultured into the same medium (and grown to mid-log phase). Equal volumes of cultures were taken, and cells were harvested by centrifugation. The cell pellets were resuspended in a volume of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (20) determined by dividing the optical density at 600 nm (OD₆₀₀) by 3 (which normalized the number of cells per milliliter for all samples). Cells were lysed by boiling the preparations for 10 min, and 15-μl samples were electrophoresed as described previously (20) on an SDS-9% polyacrylamide gel, transferred to nitrocellulose membranes, and subjected to Western blot analysis. MBP and LamB antisera were used at 1:5,000 dilutions. All antibodies were added simultaneously.

Western blot analysis to visualize LamB folding intermediates was performed as described above, but with the following differences. Cells were gently lysed as previously described by Misra et al. (26). Samples were resuspended in sample buffer containing 3% (wt/vol) SDS, 10% (vol/vol) glycerol, and 5% (vol/vol) β-mercaptoethanol in 70 mM Tris-HCl (pH 6.8). Samples were aliquoted into separate tubes and heated to the appropriate temperature. SDS-PAGE analysis was performed using 9% polyacrylamide, and gels were electrophoresed at low voltage (less than 60 V) so as not to denature folding intermediates of LamB. Following SDS-PAGE, samples were transferred to Immobilon (Millipore) as described by the manufacturer, and Western blot analysis was performed as described above.

Crude outer membrane preparations and protein sequencing.

Crude outer membranes were prepared with some variations on a previously published protocol (28). Strains were grown to saturation in maltose minimal medium at 30°C. Cells were harvested by centrifugation at room temperature for 15 min at 3,000 rpm in a Sorvall SM24 rotor. The supernatant was removed, and cells were placed on dry ice-ethanol for 4 min. The pellets were thawed and resuspended in 200 μl of 20% (wt/vol) sucrose in 30 mM Tris-HCl (pH 8.0). After the addition of 50 μl of lysozyme (5 mg/ml in 100 mM EDTA [pH 7.5]), the cell suspension was placed on ice for 30 min. Cells were lysed by adding 3 ml of 3 mM EDTA (pH 7.5) and by 1 min of sonication (in 15-s pulses). Unlysed cells were removed by centrifugation for 15 min at 3,000 rpm, and the supernatant was centrifuged at 15,000 rpm in a Sorvall SM24 rotor for 60 min to pellet the crude outer membranes. The pellets were resuspended overnight at −20°C in 10 mM Tris (pH 6.7) and 100 μl of sample buffer (20). The samples were analyzed by SDS-PAGE as described previously (20). Samples were transferred to Immobilon (Millipore) and stained with Coomassie brilliant blue R-250 (Sigma) in 1% acetic acid and 40% methanol. Membranes were destained in 50% methanol, and the appropriate bands were excised for N-terminal sequencing by Edman degradation. Sequencing analysis was performed by the Princeton University Synthesis/Sequencing facility.

Pulse-labeling and immunoprecipitations.

Pulse-labeling was performed as described previously (20). For native immunoprecipitation of LamB folding intermediates, the labeled cells were lysed as described previously (26) and immunoprecipitated using antisera to LamB monomer, LamB trimer, and MBP followed by S. aureus cells. Samples were resuspended in sample buffer containing 3% (wt/vol) SDS, 10% (vol/vol) glycerol, and 5% (vol/vol) β-mercaptoethanol in 70 mM Tris-HCl (pH 6.8). Resuspended samples were solubilized at room temperature for 3 h, at which point S. aureus cells were removed by centrifugation. Samples were aliquoted into separate tubes before heating.

Cell fractionations.

Cell fractionations were done according to the method of Nikaido (30) with some changes. Cells were grown overnight in glycerol minimal medium. Five-milliliter overnight cultures were subcultured into 100 ml of fresh glycerol minimal medium and were grown to an OD₆₀₀ of approximately 0.4, at which point LamB synthesis was induced with 0.4% (wt/vol) maltose. Cultures were then grown to an OD₆₀₀ of 1.0. First, periplasmic contents were collected by spheroplasting as follows. Cells were pelleted for 5 min at 5,000 rpm in a Sorvall SM24 rotor followed by a wash in 1/10 volume of glycerol minimal medium and subsequent centrifugation as described above to pellet the cells again. At this point in the procedure, all components of the experiment were kept on ice. Harvested cells were rapidly resuspended in 6 ml of cold 0.75 M Tris-HCl (pH 7.8). Three hundred microliters of lysozyme (2 mg/ml) was immediately added, and the suspension was incubated on ice for 2 min. Twelve milliliters of cold 1.5 mM NaEDTA (pH 7.5) was added at 1 ml/min. Following this, 180 μl of phenylmethylsulfonyl fluoride (100 mM) prepared in ethanol was added, and the cells were incubated on ice for 1 h with occasional swirling. Spheroplasts were pelleted at 12,500 rpm in a Sorvall SM24 rotor for 5 min. The resulting supernatant contained the periplasmic contents. To obtain membranes, the spheroplast pellet was resuspended in 4 ml of 50 mM Tris-HCl (pH 7.5) supplemented with DNase and RNase at 20 μg/ml, as well as the following protease inhibitors: 4 μl of aprotinin, 20 μl of 100 mM phenylmethylsulfonyl fluoride, and 1 μl of pepstatin (all purchased from Sigma). The spheroplast suspension was passed through a French pressure cell three times at 10,000 lb/in² in order to break open the spheroplasts. Unbroken cells and debris were collected by multiple centrifugation steps at 1,000 × g for 10 min at 4°C. This step was repeated until a pellet was no longer visible. To ensure that all unlysed cell and debris were removed, one additional centrifugation step was performed. EDTA was added to the membrane suspension to a 1 mM concentration, and lysozyme was added to 0.1 mg/ml in order to disassociate proteins from the peptidoglycan that would otherwise cofractionate with the outer membrane. The lysate was incubated on ice for 30 min with occasional swirling. The final lysate was added to the top of a preliminary sucrose gradient containing 1.0 ml of 25% (wt/wt) sucrose layered over 0.3 ml of 65% (wt/wt) sucrose. Samples were centrifuged at 55,000 rpm, 4°C (SW55 rotor) in a Beckman ultracentrifuge for 2 h. This step separates cytoplasmic contents from debris and membranes. The top 3 ml of the samples was saved as cytoplasm, the next 1 ml was discarded as cellular debris, and the bottom 1 ml of enriched membranes was collected from the bottom of the tube. This membrane sample was mixed with 1.4 ml of EDTA (5 mM) and was loaded on a secondary sucrose gradient with the following concentrations of sucrose from bottom to top: 0.5 ml of 65% (wt/wt) sucrose, 0.5 ml of 55% (wt/wt) sucrose, 1 ml of 50% (wt/wt) sucrose, 2 ml of 45% (wt/wt) sucrose, 2 ml of 40% (wt/wt) sucrose, 2 ml of 35% (wt/wt) sucrose, and 1.5 ml of 30% (wt/wt) sucrose. Gradients were centrifuged for 17 h at 36,000 rpm in a Beckman Ultracentrifuge (SS41 rotor) (4°C). Collected fractions were subjected to SDS-PAGE followed by Coomassie staining, and the appropriate fractions were used for Western blot analysis as described above.

Nonreducing SDS-PAGE.

Nonreducing conditions were used to study disulfide bond formation. Cells were grown overnight in glycerol minimal medium and then subcultured 1:50 into the same media. At an OD₆₀₀ between 0.30 and 0.35, cells were induced with 0.4% (wt/vol) maltose for approximately 1 h. Cells were gently lysed following the previously described protocol (26) except that 50 mM iodacetamide (IAA) was added to the SDS lysis buffer. Samples were resuspended in SDS loading buffer (20) containing either 2 mM IAA or, as a control, 360 mM β-mercaptoethanol. Samples were aliquoted, heated to temperatures of 37, 55, 70, or 100°C, and subjected to SDS-9% PAGE as previously described (20). To prevent contamination with β-mercaptoethanol, at least one lane was left empty between reduced and nonreduced samples.

RESULTS

Rationale and mutant isolation.

lamBA23DA25Y and lamBA23YA25Y (lamBDY and lamBYY, respectively; see Table 1 for all mutant abbreviations) specify a LamB protein that is effectively tethered to the inner membrane by its signal sequence. The consequences of these mutations are the inability of LamB to function as both a maltodextrin pore and a bacteriophage λ receptor (8). We sought suppressors of lamBDY and lamBYY that would restore the function of LamB. Suppressors were obtained by plating 100-μl aliquots of overnight cultures grown in LB onto maltodextrin minimal agar. After several days of incubation at 30°C, mutant colonies were purified onto the same media. Those mutants that were able to utilize maltodextrins (Dex⁺) as a sole carbon source were analyzed further.

TABLE 1.

LamB mutant genotype and protein abbreviations

Mutant genotype	Abbreviated genotype	Mutant protein
lamBA23DA25Y	lamBDY	LamBDY
lamBA23YA25Y	lamBYY	LamBYY
lamBR6LA23DA25Y	lamBDY6L	LamBDY6L
lamBM19TA23DA25Y	lamBDY19T	LamBDY19T
lamBR6CA23YA25Y	lamBYY6C	LamBYY6C
lamBR6SA23YA25Y	lamBYY6S	LamBYY6S

Mutant identification.

The suppressor mutations were mapped to lamB as previously described (6). Briefly, P1 lysates grown on each of the six suppressors that were isolated as well as the parent strain were used to transduce strain NT1001 to Dex⁺. NT1001 carries a deletion (malBΔ1) extending from the 3′ end of malK through the 5′ end of lamB. Therefore, only suppressors carrying functional lamB are able to correct the Dex⁻ phenotype of the malBΔ1 strain. All six suppressors were able to restore a Dex⁺ phenotype to NT1001, at least to some extent. DNA sequence analysis revealed that all six suppressors were additional single base pair substitutions that further alter the signal sequence of the tethered LamB. The suppressors were of two different types (Fig. 1). One mutation changed a methionine to a threonine at position 19 of the signal sequence. The five remaining suppressors all changed a positively charged arginine at position six of the signal sequence to an uncharged amino acid—serine, cysteine (two isolates), or leucine (two isolates). The mutation that replaced an arginine with a serine at position six was previously identified and shown to result in the formation of a stable hairpin structure in the lamB transcript, rendering the Shine-Dalgarno sequence inaccessible to ribosomes (19). The resulting inefficient translation yields low levels of LamB.

FIG. 1.

Suppressors of lamBDY and lamBYY. The amino acid sequence of the wild-type and each of the mutant LamB signal sequences are shown. The signal sequence cleavage site of wild-type LamB is indicated by the arrow. The residue number is listed above the appropriate amino acid, and the mutated amino acid residues are shown in bold.

Suppressors of lamBDY and lamBYY restore function as porins and as λ receptors.

Phenotypes of the suppressors of tethered lamB were analyzed in several different ways. First, LamB function as a porin was determined by scoring colony color on maltodextrin MacConkey agar. Only cells carrying functional LamB will be able to utilize maltodextrins provided in the medium. As expected, cells carrying wild-type lamB were able to grow with maltodextrins as the sole carbon source and thus produce red colonies on maltodextrin MacConkey agar (Table 2). In contrast, cells producing tethered LamB (lamBDY and lamBYY) were unable to grow on maltodextrins and produced white colonies on maltodextrin MacConkey agar (Table 2). Mutants carrying lamBDY19T behaved identically to cells carrying wild-type lamB when plated on maltodextrin MacConkey agar, producing red colonies (Table 2). In contrast, two of the mutants carrying the charge changes, the lamBDY6L or lamBYY6C mutants, gave rise to pink colonies, a phenotype intermediate between those of the wild type and the parent strains (Table 2).

TABLE 2.

Maltodextrin utilization and λ sensitivity phenotypes of lamBDY and lamBYY suppressors

Relevant genotype	Dex phenotypea	λ phenotypeb
lamB+	Red	S
lamBDY	White	R
lamBYY	White	R
lamBDY19T	Red	S
lamBDY6L	Pink	S/R
lamBYY6C	Pink	S/R
lamBYY6S	White	R

^aDex phenotype was characterized by scoring colony color on dextrin MacConkey agar. Red, Dex⁺; pink, Dex^+/−; White, Dex⁻.

^bλ phenotype was analyzed by cross-streaking each strain against λ_vir. S, sensitive; S/R, slightly resistant; R, resistant.

We also tested each of the suppressor mutants as well as lamBDY and lamBYY mutants for growth on purified maltodextrins of various lengths. lamB wild-type cells and the suppressor mutants were all able to grow to some extent on maltodextrins with seven or less glucose residues. Mutants carrying tethered LamB were unable to grow on these maltodextrins (data not shown). These results indicate that the LamB pore encoded by the suppressor alleles is able to transport sugars that are the same size as those transported by wild-type LamB.

Similar results were obtained with sensitivity to bacteriophage λ (Table 2). Susceptibility to λ was determined by cross-streaking each strain against λ_vir. Cells producing wild-type LamB are sensitive to λ. In contrast, cells producing the tethered LamB are resistant to λ. LamBDY19T-producing cells were phenotypically identical to those producing wild-type LamB. These mutants are λ sensitive. In contrast, LamBDY6L- and LamBYY6C-producing cells were slightly resistant to λ. The LamBYY6S strain grew poorly on maltodextrins as a carbon source and was resistant to λ, as might be expected due to the decreased translation previously described (19). The above observations suggested that functional LamB is present, albeit at different levels, in each of the suppressor strains isolated.

Suppressors of lamBDY and lamBYY produce stable LamB proteins.

We initially examined the LamB steady-state protein levels by Western blot analysis using antibodies to LamB monomer. Our results not only gave us insight into the LamB levels in each strain but also into the molecular weight of the major species of LamB protein present. Cells carrying lamBDY19T produce a LamB protein that appears equivalent to that from wild-type cells, both at steady-state levels and in molecular weight (Fig. 2). In contrast, lamBDY6L and lamBYY6C mutants produce steady-state levels of LamB that are similar to those for wild-type cells, but the molecular weight of the LamB protein is equivalent to that of the tethered LamBDY and LamBYY (Fig. 2). The LamB protein levels are quite low in the lamBYY6S mutant. Again, this is expected due to the reduced translation in this mutant. The molecular weight of LamBYY6S is equal to that of tethered LamBDY and LamBYY because the signal sequence is not cleaved. Additionally, levels of tethered LamB are low because the mutant proteins are unfolded in the periplasm and consequently degraded by periplasmic proteases.

FIG. 2.

Steady-state levels of LamB are increased relative to those of tethered mutants in strains carrying suppressors, with the exception of LamBYY6S. Strains were grown to mid-log phase in LB medium. Western analysis was done as already described using antibodies to LamB monomer, LamB trimer, and MBP. The LamB trimer antibody recognizes OmpA and thus serves as an internal loading control.

Suppressors of lamBDY do not restore wild-type signal sequence processing.

Crude outer membranes were prepared from each suppressor strain, and protein profiles of these extracts were analyzed by SDS-PAGE and Coomassie staining (Fig. 3A). Cells carrying suppressors of tethered LamB exhibit an induced PspA stress response, as do cells producing tethered LamBDY and LamBYY (Fig. 3A). The reasons for this induction are not clear, but it appears that the suppressors that we have isolated do not entirely relieve the stresses caused by the cleavage site mutations.

FIG. 3.

(A) Crude outer membrane preparations of LamBDY suppressors. LamB and PspA (identified using anti-PspA antibody; data not shown) are indicated by the arrows, and the asterisk marks the location of the major outer membrane proteins OmpF, OmpC, and OmpA. (B) Suppressors of tethered LamB retain either part or all of the signal sequence in the outer membrane. N-terminal protein sequencing was done on LamB obtained from the crude outer membranes shown in panel A. Mature LamB protein is indicated by the stippled bar, and the LamB signal sequence is indicated by the white bar. The positions of signal sequence cleavage are indicated by the arrows.

The N-terminal amino acid sequence of LamB protein recovered from the crude outer membrane preparations was determined (Fig. 3B). As expected, wild-type LamB was present in the outer membrane as only the mature protein. LamBDY19T exists in the outer membrane with the last four amino acids of the signal sequence at its N terminus (Fig. 3B). Clearly, this mutation creates an alternate processing site. We might expect such a result because the replacement of a bulky methionine with a smaller threonine produces a sequence (TSA) very close to the consensus site (AXA) recognized by signal peptidase (41). Levels of this mutant appeared to be quite low in the outer membrane. Upon further analysis, we determined that processing of this mutant is more than four times slower than that of wild-type LamB (data not shown). We hypothesize that slowed processing leads to unfolded LamB in the periplasm for an extended period of time, resulting in degradation of a significant fraction of the protein by periplasmic proteases.

Much more surprising are the results of protein sequencing of the charge change mutant. We found that LamBDY6L that was recovered from outer membrane preparations retained the entire signal sequence of LamB (Fig. 3B). Despite the presence of this 25-amino-acid extension, LamB remained functional and stable in the outer membrane.

A novel LamB species is present in cells carrying lamBDY6L.

As discussed in the introduction, LamBDY and LamBYY are tethered to the inner membrane where they are degraded. Several types of suppressors of the toxicity caused by this tethering have been isolated, but they have resulted in nonfunctional LamB localized either to the inner or outer membrane (11). We sought to determine whether the charge change suppressors of lamBDY isolated in this study could form stable trimers in the outer membrane despite the presence of the entire signal sequence. To investigate the properties of LamB in each of the suppressor mutants further, we performed Western blot analysis in which cells were gently lysed to allow for the recovery of LamB folding intermediates (Fig. 4). Briefly, wild-type cells and those carrying the charge change mutation were lysed as previously described to prevent the dissociation of higher-molecular-weight intermediates and the LamB trimer (26). Samples were resuspended in sample buffer and heated to 37, 55, 70, or 100°C. After SDS-PAGE analysis, samples were transferred to a polyvinylidene difluoride membrane and then exposed to antibodies recognizing LamB monomer, LamB trimer, and MBP (26). As expected, wild-type LamB trimer dissociates completely into unfolded monomer above 70°C (Fig. 4). Surprisingly, despite the functionality of LamBDY6L, a protein species corresponding to wild-type trimer does not exist. Rather, a novel species is observed in samples from these charge change mutants (Fig. 4). This species denatures into LamB monomer at temperatures between 37 and 55°C. In addition, we are able to observe folded LamB monomer in the charge change mutant background at 37°C (Fig. 4). This result is intriguing because folded LamB monomer has not been observed in all strain backgrounds. Rouviere and Gross reported the existence of the folded monomer intermediate of LamB in strain MC1061 (36). The data presented in Fig. 4 suggest that we may have slowed LamB assembly in the charge change mutants, thus increasing the levels of LamB folding intermediates.

FIG. 4.

A novel LamB species that denatures at low temperatures is observed in the charge change suppressor. Cells were gently lysed as described in Materials and Methods. Samples were heated to either 37, 55, 70, or 100°C prior to SDS-PAGE. Western analysis was done using antibody to LamB trimer, LamB monomer, or MBP. The positions of the various LamB species and MBP are noted by the arrows. The LamB trimer antibody recognizes OmpA and thus serves as an internal loading control. OmpA is heat modifiable and migrates more slowly at 100°C. p, precursor; m, mature

LamB localizes to the outer membrane in the lamBDY6L mutant.

To clearly demonstrate the cellular localization of this species, we performed membrane fractionations. The inner and outer membranes were separated by sucrose gradient centrifugation, and the resulting fractions were analyzed by Coomassie staining. The fractions corresponding to inner and outer membranes were analyzed further by Western analysis (Fig. 5).

FIG. 5.

Folded LamBDY6L fractionates with the outer membrane. Cells were fractionated as described in Materials and Methods. Fractions were visualized on a Coomassie-stained gel. The fractions corresponding to the inner and outer membranes were subjected to Western blot analysis. Prior to SDS-PAGE, outer membrane samples were heated to either 37 or 100°C as noted above each lane. LamB folding intermediates are indicated by the arrows. The LamB trimer antibody recognizes OmpA and thus serves as an internal loading control.

In order to detect LamB folding intermediates, which are present at low levels, the gel shown in Fig. 5 was purposely overexposed. Consequently, proteins such as OmpA that are present at very high levels appear in all fractions. In control experiments we showed that the outer membrane protein OmpF and an inner membrane protein of unknown function of approximately 55 kDa (5) fractionate correctly (data not shown).

In wild-type cells, the majority of LamB localizes in the outer membrane fractions (Fig. 5). Similarly, we found that the novel species seen in the lamBDY6L mutant also localized to the outer membrane (Fig. 5).

The location of the folded LamB monomer has not been reported. Our fractionation experiments show that folded LamB monomer localized to the outer membrane (Fig. 5). We also fractionated strain MC1061 for which the folded monomer has been clearly demonstrated (36). Consistent with our observations above, the folded monomer of LamB localized to the outer membrane in MC1061, although the levels of this species in a wild-type strain are clearly lower than those observed in the charge change mutant (Fig. 5). We are unable to determine if the folded monomer is actually inserted into the outer membrane or whether it is associated with the membrane at its periplasmic face. Again, we propose that the lamBDY6L mutant has resulted in slowed LamB assembly that has allowed clear visualization of the folded monomer in fractionation experiments.

LamB forms multimers in the lamBYY6C mutant.

In order to understand the identity of the novel species observed with the lamBDY6L mutant, we took advantage of another suppressor that was isolated in the study and found to behave identically to lamBDY6L in phenotypic assays and Western blot analysis. This mutant, lamBYY6C, substitutes the highly charged arginine at position six of the signal sequence with an uncharged cysteine residue. We hypothesized that the mutants that retain the signal sequence but are functional are positioned as multimers in the outer membrane with their signal sequences clustered together in a bundle such that the LamB pore is not obstructed by them. This being the case, we proposed that two of the cysteine mutant signal sequences would have the potential to form a disulfide bond with each other.

To test the possibility that the charge change mutants have the ability to form multimers, we prepared lysates of the lamBYY6C mutant using the gentle lysis procedure described above, with the exception that IAA was added to the lysis buffer. IAA interacts with free sulfhydryl groups to prevent the formation of disulfide bonds by oxidation during sample handling. Thus, disulfide bonds observed must have been formed in vivo. Following cell lysis, we incubated samples at various temperatures under both reducing (in the presence of β-mercaptoethanol) and nonreducing (in the presence of IAA) conditions, and we observed LamB by Western blot analysis (Fig. 6). When comparing the data in Fig. 4 and Fig. 6 (left panel), it can be seen that lamBYY6C mutants prepared under reducing conditions behaved identically to lamBDY6L mutants prepared under the same conditions. At the low temperature (37°C), the novel band observed for the lamBDY6L mutant was also present for the lamBYY6C mutant and, similarly, this band denatures to LamB monomer at or below 55°C. In contrast, when the lamBYY6C mutant was prepared and electrophoresed under nonreducing conditions, the novel band was stabilized and persisted until temperatures exceeded 70°C (Fig. 6). Temperatures at or above 55°C did result in the accumulation of some LamB monomer and a species of a higher molecular weight (Fig. 6). At 100°C, the novel species denatured completely, and LamB monomer and a species that corresponds to a molecular mass of approximately 90 kDa appeared (Fig. 6). This molecular size is close to that of two LamB monomers, indicating that under nonreducing conditions, LamBYY6C is able to form a disulfide-bonded dimer. We also note that a small amount of LamB dimer was present at 37°C. We believe that this is due to inadvertent heating that may have occurred during lysis (specifically centrifugation) of the samples. At 100°C, an additional protein with a slightly faster mobility than the LamB dimer was present (Fig. 6). We suggest that in addition to the intermolecular disulfide bond that forms in the lamBYY6C mutant, this species also contains an intramolecular disulfide bond involving one or both of the cysteines that are located in one of the surface loops of LamB. Another possibility is that the lower-molecular-weight protein is LamB monomer that is also disulfide bonded to another protein. Intramolecular disulfide bonding between the two cysteines on surface loop 1 of LamB has been reported (22, 23). We have not yet investigated these possibilities further. The LamB monomers that accumulate at temperatures above 37°C could be components of the trimer that were not participants in disulfide bonding. Therefore, these experiments suggest that LamB mutants that retain their entire signal sequence are, in fact, able to form multimers.

FIG. 6.

LamB charge change mutants from multimers. Cells carrying lamBYY6C were gently lysed and treated with either β-mercaptoethanol or IAA. Samples were heated to the temperatures noted above each lane. Proteins were electrophoresed and analyzed by Western blot analysis. Antibodies to LamB trimer, LamB monomer, and MBP were used. The various folded and unfolded LamB proteins are indicated by the arrows. OmpA, recognized by LamB trimer antibody, serves as an internal loading control. OmpA is heat modifiable and migrates more slowly at 100°C

lamBDY6L slows the formation of LamB folding intermediates.

We hypothesized that in the charge change mutant, assembly of LamB might be slowed due to the presence of the entire signal sequence. To test this idea, we followed the kinetics of LamB trimerization in both wild-type cells and the lamBDY6L mutant. Figure 7 shows a pulse-chase experiment called a trimer assay (26). Cells were labeled with [³⁵S]methionine followed by a native immunoprecipitation with antibodies that recognize the monomer form of LamB, the trimer, and MBP as a loading control (26). Following immunoprecipitation, samples were resuspended in sample buffer and heated to either 37 or 70°C. In wild-type cells, heating to 70°C stripped the lipopolysaccharide (LPS) from the trimer and denatured the LamB assembly intermediates. SDS-PAGE analysis allowed the separation of the trimer from the denatured monomer LamB. At low temperatures, wild-type LamB trimers chased to LPS-associated trimer by approximately 2′ postchase (Fig. 7, top). At 70°C, the trimers were dissociated from LPS and thus had a faster mobility (Fig. 7, bottom).

FIG. 7.

A novel species recognized by LamB trimer antibody is present in the charge change mutants. Cells carrying either wild-type lamB or lamBR6LA23DA23Y were subjected to pulse-chase labeling followed by native immunoprecipitation with antibodies recognizing unfolded LamB monomer, folded LamB, and MBP (as described in the Materials and Methods). Shown above are the autoradiography films of SDS-PAGE gels. Increasing chase times are indicated by the triangles below each gel. The times were as follows: 30 s, 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 30 min, and 60 min. Prior to electrophoresis, samples were heated to either 37°C (left) or 70°C (right). Positions of various LamB species and MBP are noted by the arrows. p, precursor; m, mature.

We noticed several differences in the formation of LamB folding intermediates with the lamBDY6L mutant. First, the transition from LamB monomer to a higher-molecular-weight species was slowed—monomer was present as late as 10 min postchase (Fig. 7, top). As mentioned above, in certain strain backgrounds a folded monomer species has been visualized in a trimer assay (36). In the MC4100 background we are working with, the presence of folded LamB monomer is often not observed. However, we were able to visualize the folded monomer species of LamB in the lamBDY6L mutant background (Fig. 7, top). The trimer assay results show that, as with the transition of unfolded monomer into a higher-molecular-weight species, the disappearance of folded monomer was slowed in the charge change mutant (Fig. 7, top). Consistent with previous data, the folded monomer in our experiments melted at 70°C (Fig. 7, bottom).

The results of the kinetic experiment in Fig. 7 demonstrate that the novel LamB species observed in Western blot analysis of lamBDY6L mutants was also present in the trimer assays. The folded monomer LamB chased into this higher-molecular-weight species, which is distinct from wild-type LamB trimer (Fig. 7, top). The functionality of LamB in the charge change mutant background and the kinetics of appearance of this novel species suggest that it is a LamB trimer.

DISCUSSION

We report here the isolation of suppressors of tethered lamBDY and lamBYY.

The goal of our work has been to understand the targeting and folding of a class of outer membrane proteins collectively termed the β-barrels. As a model, we have been studying the maltoporin LamB. Efforts to isolate targeting and folding factors genetically have been generally unsuccessful, possibly due to functional redundancy within the periplasm of E. coli. To exaggerate the defect caused by null mutations in proteins of redundant function, attempts were made to isolate lamB mutants slowed in targeting and/or folding. In particular, mutations that hinder cleavage of the LamB signal sequence by signal peptidase were constructed. These mutants resulted in leaky phenotypes which eventually targeted mutant LamB (6) or in LamB molecules which were completely blocked in signal sequence processing, resulting in the tethering of the protein to the inner membrane by its signal sequence (10). The former LamB mutants are targeted as efficiently as wild-type LamB (6), and the latter mutations resulted in LamB protein that was no longer functional because it was tethered to the inner membrane (10).

In this study, we have isolated functional suppressors of lamBDY and lamBYY. The two classes of suppressors are distinct. We have shown that one mechanism of lamBDY suppression is the creation of a novel signal sequence cleavage site. Another, distinct mechanism is the relief of a strong positive charge at the amino terminus of the signal sequence. Interestingly, others have predicted that inner membrane proteins are positioned in the membrane such that positively charged residues located in the cytoplasmic portion of the signal sequence serve to anchor transmembrane domains (4). The charge change mutants that we have isolated offer further support for this “positive inside rule.” The arginine that is found in the wild-type LamB signal sequence serves as an anchor that retains the signal sequence in the inner membrane. When this arginine is changed to an uncharged amino acid, such as leucine or cysteine, the strength of the anchor is reduced, allowing the signal sequence to slip through the inner membrane. Once LamB is released from the inner membrane, it can localize to the outer membrane as a multimer.

The lack of signal sequence cleavage in the charge change mutant is not without consequences. We have shown that the charge change mutants are slowed in assembly. This may result from the mechanism that mutant LamB utilizes to escape from the inner membrane. It is tempting to speculate that this mechanism involves periplasmic folding factors that would normally be involved in the proper folding of wild-type LamB. In this case, the partial folding of LamB catalyzed by these factors may provide the energy necessary to pull the signal sequence through the inner membrane.

Regardless of the mechanism by which the charge change LamB mutants are released from the inner membrane, the slowed assembly has allowed us to follow the folding of LamB and to visualize folding intermediates that are not seen in cells carrying wild-type LamB. In certain strain backgrounds, the existence of a folded monomer intermediate of LamB has been observed (36). We have not been able to observe the folded monomer in the MC4100 wild-type background. However, in the charge change mutants, slow assembly reveals a form of LamB that, like the folded monomer previously reported, melts into a higher-molecular-weight form of LamB. In the case of the charge change mutant, the folded monomer melts into a precursor LamB owing to the presence of the signal sequence.

Although the existence of the folded monomer has been shown using biochemical approaches (36), the cellular localization of this folding intermediate has not been elucidated. We have shown that the folded monomer localizes to the outer membrane. We cannot determine whether the folded monomer is positioned within the outer membrane or whether it is associated in some other fashion. Nonetheless, this is an intriguing result because it suggests that the LamB trimers assemble in the outer membrane. The idea that LamB trimerization occurs at the outer membrane rather than in the periplasm has been proposed in previous studies (26).

It has proven difficult to determine whether the novel-molecular-weight species observed in the charge change mutants is actually LamB trimer. It is impossible to predict how a LamB trimer with three additional hydrophobic sequences (the signal sequences) will run in SDS-PAGE. Because strains carrying these mutations are LamB⁺, we favor the proposal that the novel band is a LamB trimer that, due to its increased hydrophobicity, binds more SDS and thus runs faster through the gel than wild-type LamB trimer. We do know that LamB forms a multimer of at least two monomers in the outer membrane. Proof for this lies in the fact that when run under nonreducing conditions, the novel band is stabilized in the cysteine charge change mutant. In addition, this species melts into a protein that has a molecular weight equal to two LamB monomers. The fact that we also observe a protein that has a molecular weight equal to that of the LamB monomer in these preparations may provide further evidence for trimer formation. Obviously, only two LamB monomers can participate in dimer formation by disulfide bonding.

An alternative explanation for the novel species is that it is a LamB dimer. There has been little evidence for the existence of β-barrel dimers even though dimers must be an obligatory intermediate in trimer formation. Two studies have suggested that OmpF and OmpC form dimers that are loosely associated with the outer membrane (33, 35). However, it seems unlikely that a β-barrel dimer would be functional in the outer membrane. This would require proteins like LamB to have very large hydrophilic surfaces contacting the very hydrophobic environment of the membrane, and the stability of such a protein in the outer membrane is questionable.

We favor the possibility that LamB can form stable trimers even with the signal sequence attached. Interestingly, a precedent for such trimers does exist. The sucrose-specific porin (ScrY) of Salmonella enterica serovar Typhimurium has been shown to have high structural homology to LamB (18). The major difference between ScrY and LamB lies in a periplasmic 70-amino-acid extension at the N terminus of the ScrY protein (14). Both LamB and ScrY possess aromatic residues within the pore of the β-barrel that have been postulated to form a slide through which sugars could pass en route to the periplasm (14, 38). The N-terminal extension of ScrY is thought to be an extension of this sugar slide (14). We have shown that a LamB mutant retaining its signal sequence is functional, but not as functional as the wild-type LamB porin. Therefore, it would seem that an N-terminal extension such as that seen in ScrY is not necessarily desirable in other bacterial porins, such as LamB.

In this study, we have isolated a functional suppressor of tethered LamB. As mentioned above, lamBDY was originally constructed to slow down the assembly of LamB in the outer membrane. Instead, however, these mutations resulted in a LamB protein that is not released from the inner membrane. We have shown that the charge change mutant relieves the tethering and allows assembly, albeit slowed, in the outer membrane. The mechanism by which LamBDY6L and LamBYY6C are released from the inner membrane is intriguing. It is possible that these mutants leave the inner membrane spontaneously. Alternatively, the LamB signal sequence may be pulled out of the membrane by periplasmic folding factors.