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Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
Received 29 April 2002/ Accepted 16 September 2002
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 ß-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 ß-barrel proteins is less well understood. These proteins include the outer membrane porins PhoE, OmpF, OmpC, and LamB. Our model for studying ß-barrel proteins is LamB, the porin utilized for the import of maltodextrins and the cell's receptor for bacteriophage 
. The structure of the LamB porin has been elucidated, and it demonstrates that LamB forms an 18-stranded ß-barrel in which each strand is amphipathic with alternating hydrophobic and hydrophilic amino acid residues (38).
As with other extracytoplasmic proteins, the ß-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 
-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, ß-barrel proteins, such as those mentioned above, are targeted very efficiently to the outer membrane (for a review, see references 12 and 31). Although ß-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). ß-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 receptor. Cells carrying these mutations are Dex- and 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.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bacterial strains and microbiological techniques.
The bacterial strains used in this study are derivatives of E. coli K-12 strain MC4100 [F-araD139 
(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) ß-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) ß-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 x 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 ß-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 ß-mercaptoethanol, at least one lane was left empty between reduced and nonreduced samples.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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1) 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 malB1 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.
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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).
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Similar results were obtained with sensitivity to bacteriophage (Table 2). Susceptibility to was determined by cross-streaking each strain against vir. Cells producing wild-type LamB are sensitive to . In contrast, cells producing the tethered LamB are resistant to . LamBDY19T-producing cells were phenotypically identical to those producing wild-type LamB. These mutants are sensitive. In contrast, LamBDY6L- and LamBYY6C-producing cells were slightly resistant to . The LamBYY6S strain grew poorly on maltodextrins as a carbon source and was resistant to , 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.
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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.
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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 ß-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.
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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.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 ß-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 ß-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 ß-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.
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ACKNOWLEDGMENTS |
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A.R.D. was supported by an NIH departmental training grant (GM 07388); T.J.S. was supported by an NIGMS MERIT grant (GM 34821).
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FOOTNOTES |
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REFERENCES |
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