signal sequence mutations as tools for the characterization ...lipoproteins are complexed with a...

JOURNAL OF BACTERIOLOGY, Dec. 2002, p. 6918–6928 Vol. 184, No. 240021-9193/02/$04.00�0 DOI: 10.1128/JB.184.24.6918–6928.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Signal Sequence Mutations as Tools for the Characterizationof LamB Folding Intermediates

Amy Rizzitello Duguay and Thomas J. Silhavy*Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

Received 29 April 2002/Accepted 16 September 2002

lamBA23DA25Y and lamBA23YA25Y tether LamB to the inner membrane by blocking signal sequenceprocessing. We isolated suppressors of lamBA23DA25Y and lamBA23YA25Y, all of which mapped within theLamB signal sequence. Most interesting were mutations that changed an amino acid with a strong positivecharge to an amino acid with no charge. Further characterization of two such suppressors revealed that theyproduce 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 propertiesdifferent from those of wild-type LamB trimer. Because assembly of mutant LamB is slowed, these mutationsprovide useful tools for the characterization of LamB folding intermediates.

In Escherichia coli, extracytoplasmic proteins must be tar-geted to three distinct compartments—the periplasm and theinner and outer membranes. All proteins in E. coli are initiallysynthesized in the cytoplasm, but the path that they then followdepends upon their ultimate cellular destination. Many pro-teins bound for extracytoplasmic locations are synthesized withan N-terminal signal sequence that directs them to the generalsecretion machinery at the inner membrane (for a review, seereference 12).

Two models dominate studies of outer membrane proteintargeting. The first of these is the Bayer’s junction model,which proposes that proteins travel to the outer membranethrough zones of adhesion between the inner and outer mem-branes (1). The second model, known as the periplasmic in-termediate model, predicts that outer membrane proteins passthrough the secretion machinery at the inner membrane andinto the periplasm before becoming localized to the outermembrane (31). The periplasmic intermediate model mightsuggest that proteins transiting through the periplasm are metby factors that aid in their folding. Indeed, several differentgroups of periplasmic folding factors have been identified.They include proteins involved in the formation and isomer-ization 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 proteinsin the outer membrane: surface organelles such as pili, lipopro-teins, and the �-barrel proteins. The targeting of many pili isknown to involve the periplasmic chaperone/usher pathway(37). Recently, the targeting of outer membrane lipoproteinshas been elucidated (24, 25). Lipoproteins are complexed witha periplasmic protein, LolA, which carries them to the outermembrane, where they are met by the outer membrane-asso-ciated component, LolB. LolB is intimately involved with theincorporation of lipoprotein into the outer membrane (24, 25).

The folding and targeting of the �-barrel proteins is less wellunderstood. These proteins include the outer membrane por-

ins PhoE, OmpF, OmpC, and LamB. Our model for studying�-barrel proteins is LamB, the porin utilized for the import ofmaltodextrins and the cell’s receptor for bacteriophage �. Thestructure of the LamB porin has been elucidated, and it dem-onstrates that LamB forms an 18-stranded �-barrel in whicheach strand is amphipathic with alternating hydrophobic andhydrophilic amino acid residues (38).

As with other extracytoplasmic proteins, the �-barrel pro-teins are initially synthesized in the cytoplasm with an N-ter-minal signal sequence (15). The LamB signal sequence consistsof three major regions (42). At the N terminus, there are twobasic amino acids. Following these positively charged residuesis a stretch of hydrophobic amino acids that assume an �-he-lical structure when the signal sequence is looped into theinner membrane (16). At the C-terminal end of the signalsequence are one or more helix-breaking residues and a con-sensus site for cleavage by signal peptidase. The consensuscleavage site generally consists of any amino acid bound onboth sides by alanine (A-X-A) (42). Although variations in thesignal sequence are tolerable, replacement of either of thesetwo alanines by a bulkier amino acid results in loss of cleavageby signal peptidase (10, 17). If processing of the signal se-quence is blocked, the protein remains tethered to the innermembrane (9, 10, 17).

After signal sequence cleavage, �-barrel proteins, such asthose mentioned above, are targeted very efficiently to theouter membrane (for a review, see references 12 and 31).Although �-barrel proteins exist in the outer membrane astrimers, several folding intermediates have been described.These assembly intermediates include the mature unfoldedmonomer, folded monomer, dimer, and metastable trimer (fora review, see reference 12). �-barrel dimers have been re-ported for OmpF and OmpC (33, 35). With respect to LamB,evidence for the existence of an unfolded monomer, metasta-ble trimer, and mature trimer is clear (26, 33). Folded mono-mer intermediates of LamB have also been found in certainstrain backgrounds (36), but the cellular location of this inter-mediate has remained a mystery.

To understand how outer membrane proteins are targetedand inserted in the outer membrane, mutations that slow down

* Corresponding author. Mailing address: 310 Lewis Thomas Lab-oratory, Princeton University, Princeton, NJ 08544. Phone: (609) 258-5899. Fax: (609) 258-2957. E-mail: [email protected].

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or prevent processing and targeting of LamB have beenconstructed (6, 10). In particular, lamBA23DA25Y andlamBA23YA25Y both contain two mutations that alter the sig-nal sequence consensus cleavage site, preventing cleavage bysignal peptidase. Thus, LamB effectively becomes tethered tothe inner membrane (8). The consequences of this tetheringare threefold. First, tethered LamB is unable to function as apore for the import of maltodextrins or as a � receptor. Cellscarrying these mutations are Dex� and �R. Tethering alsoprevents LamB folding, and as a consequence, it is degraded inthe periplasm. Finally, tethered LamB is toxic to the cell, asevidenced by sensitivity to inducers such as maltose (10). Thereasons for this toxicity are unknown.

In this study we report the isolation and characterization offunctional suppressors of lamBA23DA25Y and lamBA23YA25Y.Using these mutants, we provide evidence that LamB oli-gomers are functional in the outer membrane even with theentire signal sequence still attached. Furthermore, we showthat the mutations slow LamB assembly in the outer mem-brane significantly. This crippling of LamB assembly hasenabled us to localize a key assembly intermediate in thecell.

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 supplementedwith 0.4% (wt/vol) sugars and 0.5% (vol/vol) Luria-Bertani (LB) broth. [35S]me-thionine was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, Calif.).LamB and maltose-binding protein (MBP) antisera are from our laboratorystock (26). Formalin-fixed Staphylococcus aureus used for immunoprecipitationswas purchased from CalBiochem. Enhanced chemiluminescence (ECL) Westernblotting reagents were purchased from Amersham Life Science (Piscataway,N.J.).

Bacterial strains and microbiological techniques. The bacterial strains used inthis study are derivatives of E. coli K-12 strain MC4100 [F�araD139 �(argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR] (39). Standard microbio-logical methods used for P1 transduction have been described previously (39).

Western analysis. For standard Western blot analysis, cells were grown over-night in glycerol minimal medium at 37°C. Cells were subcultured into freshglycerol minimal medium and grown to early log phase, at which point LamBsynthesis was induced with 0.4% (wt/vol) maltose for 1 to 2 h at 37°C. Alterna-tively, cells were grown to saturation in LB liquid medium and subcultured intothe same medium (and grown to mid-log phase). Equal volumes of cultures weretaken, and cells were harvested by centrifugation. The cell pellets were resus-pended in a volume of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) sample buffer (20) determined by dividing the optical density at600 nm (OD600) by 3 (which normalized the number of cells per milliliter for allsamples). Cells were lysed by boiling the preparations for 10 min, and 15-�lsamples were electrophoresed as described previously (20) on an SDS–9% poly-acrylamide gel, transferred to nitrocellulose membranes, and subjected to West-ern blot analysis. MBP and LamB antisera were used at 1:5,000 dilutions. Allantibodies were added simultaneously.

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

Crude outer membrane preparations and protein sequencing. Crude outermembranes were prepared with some variations on a previously published pro-tocol (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 wereplaced on dry ice-ethanol for 4 min. The pellets were thawed and resuspended in200 �l of 20% (wt/vol) sucrose in 30 mM Tris-HCl (pH 8.0). After the additionof 50 �l of lysozyme (5 mg/ml in 100 mM EDTA [pH 7.5]), the cell suspensionwas 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 removedby centrifugation for 15 min at 3,000 rpm, and the supernatant was centrifugedat 15,000 rpm in a Sorvall SM24 rotor for 60 min to pellet the crude outermembranes. The pellets were resuspended overnight at �20°C in 10 mM Tris(pH 6.7) and 100 �l of sample buffer (20). The samples were analyzed bySDS-PAGE as described previously (20). Samples were transferred to Immo-bilon (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 Edmandegradation. Sequencing analysis was performed by the Princeton UniversitySynthesis/Sequencing facility.

Pulse-labeling and immunoprecipitations. Pulse-labeling was performed asdescribed previously (20). For native immunoprecipitation of LamB foldingintermediates, the labeled cells were lysed as described previously (26) andimmunoprecipitated using antisera to LamB monomer, LamB trimer, and MBPfollowed by S. aureus cells. Samples were resuspended in sample buffer contain-ing 3% (wt/vol) SDS, 10% (vol/vol) glycerol, and 5% (vol/vol) �-mercaptoetha-nol in 70 mM Tris-HCl (pH 6.8). Resuspended samples were solubilized at roomtemperature for 3 h, at which point S. aureus cells were removed by centrifuga-tion. Samples were aliquoted into separate tubes before heating.

Cell fractionations. Cell fractionations were done according to the method ofNikaido (30) with some changes. Cells were grown overnight in glycerol minimalmedium. Five-milliliter overnight cultures were subcultured into 100 ml of freshglycerol minimal medium and were grown to an OD600 of approximately 0.4, atwhich point LamB synthesis was induced with 0.4% (wt/vol) maltose. Cultureswere then grown to an OD600 of 1.0. First, periplasmic contents were collectedby spheroplasting as follows. Cells were pelleted for 5 min at 5,000 rpm in aSorvall SM24 rotor followed by a wash in 1/10 volume of glycerol minimalmedium and subsequent centrifugation as described above to pellet the cellsagain. At this point in the procedure, all components of the experiment werekept on ice. Harvested cells were rapidly resuspended in 6 ml of cold 0.75 MTris-HCl (pH 7.8). Three hundred microliters of lysozyme (2 mg/ml) was imme-diately added, and the suspension was incubated on ice for 2 min. Twelvemilliliters of cold 1.5 mM NaEDTA (pH 7.5) was added at 1 ml/min. Followingthis, 180 �l of phenylmethylsulfonyl fluoride (100 mM) prepared in ethanol wasadded, 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. Theresulting 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 �g/ml, as well as the followingprotease inhibitors: 4 �l of aprotinin, 20 �l of 100 mM phenylmethylsulfonylfluoride, and 1 �l of pepstatin (all purchased from Sigma). The spheroplastsuspension 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 col-lected by multiple centrifugation steps at 1,000 � g for 10 min at 4°C. This stepwas repeated until a pellet was no longer visible. To ensure that all unlysed celland debris were removed, one additional centrifugation step was performed.EDTA was added to the membrane suspension to a 1 mM concentration, andlysozyme was added to 0.1 mg/ml in order to disassociate proteins from thepeptidoglycan that would otherwise cofractionate with the outer membrane. Thelysate was incubated on ice for 30 min with occasional swirling. The final lysatewas 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 werecentrifuged at 55,000 rpm, 4°C (SW55 rotor) in a Beckman ultracentrifuge for2 h. This step separates cytoplasmic contents from debris and membranes. Thetop 3 ml of the samples was saved as cytoplasm, the next 1 ml was discarded ascellular debris, and the bottom 1 ml of enriched membranes was collected fromthe 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 followingconcentrations of sucrose from bottom to top: 0.5 ml of 65% (wt/wt) sucrose, 0.5ml 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 of30% (wt/wt) sucrose. Gradients were centrifuged for 17 h at 36,000 rpm in aBeckman Ultracentrifuge (SS41 rotor) (4°C). Collected fractions were subjectedto SDS-PAGE followed by Coomassie staining, and the appropriate fractionswere used for Western blot analysis as described above.

Nonreducing SDS-PAGE. Nonreducing conditions were used to study disulfidebond formation. Cells were grown overnight in glycerol minimal medium and

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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 weregently lysed following the previously described protocol (26) except that 50 mMiodacetamide (IAA) was added to the SDS lysis buffer. Samples were resus-pended in SDS loading buffer (20) containing either 2 mM IAA or, as a control,360 mM �-mercaptoethanol. Samples were aliquoted, heated to temperatures of37, 55, 70, or 100°C, and subjected to SDS–9% PAGE as previously described(20). To prevent contamination with �-mercaptoethanol, at least one lane wasleft empty between reduced and nonreduced samples.

RESULTS

Rationale and mutant isolation. lamBA23DA25Y andlamBA23YA25Y (lamBDY and lamBYY, respectively; see Table1 for all mutant abbreviations) specify a LamB protein that iseffectively tethered to the inner membrane by its signal se-quence. The consequences of these mutations are the inabilityof LamB to function as both a maltodextrin pore and a bacte-riophage � receptor (8). We sought suppressors of lamBDYand lamBYY that would restore the function of LamB. Sup-pressors were obtained by plating 100-�l aliquots of overnightcultures grown in LB onto maltodextrin minimal agar. Afterseveral days of incubation at 30°C, mutant colonies were pu-rified onto the same media. Those mutants that were able toutilize maltodextrins (Dex�) as a sole carbon source wereanalyzed further.

Mutant identification. The suppressor mutations weremapped to lamB as previously described (6). Briefly, P1 lysates

grown on each of the six suppressors that were isolated as wellas the parent strain were used to transduce strain NT1001 toDex�. NT1001 carries a deletion (malB�1) extending from the3 end of malK through the 5 end of lamB. Therefore, onlysuppressors carrying functional lamB are able to correct theDex� phenotype of the malB�1 strain. All six suppressors wereable to restore a Dex� phenotype to NT1001, at least to someextent. DNA sequence analysis revealed that all six suppressorswere additional single base pair substitutions that further alterthe signal sequence of the tethered LamB. The suppressorswere of two different types (Fig. 1). One mutation changed amethionine to a threonine at position 19 of the signal se-quence. The five remaining suppressors all changed a posi-tively charged arginine at position six of the signal sequence toan uncharged amino acid—serine, cysteine (two isolates), orleucine (two isolates). The mutation that replaced an argininewith a serine at position six was previously identified andshown to result in the formation of a stable hairpin structure inthe lamB transcript, rendering the Shine-Dalgarno sequenceinaccessible to ribosomes (19). The resulting inefficient trans-lation yields low levels of LamB.

Suppressors of lamBDY and lamBYY restore function asporins and as � receptors. Phenotypes of the suppressors oftethered lamB were analyzed in several different ways. First,LamB function as a porin was determined by scoring colonycolor on maltodextrin MacConkey agar. Only cells carryingfunctional LamB will be able to utilize maltodextrins providedin the medium. As expected, cells carrying wild-type lamB wereable to grow with maltodextrins as the sole carbon source andthus produce red colonies on maltodextrin MacConkey agar(Table 2). In contrast, cells producing tethered LamB(lamBDY and lamBYY) were unable to grow on maltodextrinsand produced white colonies on maltodextrin MacConkey agar(Table 2). Mutants carrying lamBDY19T behaved identically tocells carrying wild-type lamB when plated on maltodextrinMacConkey agar, producing red colonies (Table 2). In con-trast, two of the mutants carrying the charge changes, the

FIG. 1. Suppressors of lamBDY and lamBYY. The amino acid sequence of the wild-type and each of the mutant LamB signal sequences areshown. The signal sequence cleavage site of wild-type LamB is indicated by the arrow. The residue number is listed above the appropriate aminoacid, and the mutated amino acid residues are shown in bold.

TABLE 1. LamB mutant genotype and protein abbreviations

Mutant genotype Abbreviatedgenotype Mutant protein

lamBA23DA25Y lamBDY LamBDYlamBA23YA25Y lamBYY LamBYYlamBR6LA23DA25Y lamBDY6L LamBDY6LlamBM19TA23DA25Y lamBDY19T LamBDY19TlamBR6CA23YA25Y lamBYY6C LamBYY6ClamBR6SA23YA25Y lamBYY6S LamBYY6S

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lamBDY6L or lamBYY6C mutants, gave rise to pink colonies,a phenotype intermediate between those of the wild type andthe parent strains (Table 2).

We also tested each of the suppressor mutants as well aslamBDY and lamBYY mutants for growth on purified malto-dextrins of various lengths. lamB wild-type cells and the sup-pressor mutants were all able to grow to some extent on mal-todextrins with seven or less glucose residues. Mutants carryingtethered LamB were unable to grow on these maltodextrins(data not shown). These results indicate that the LamB poreencoded by the suppressor alleles is able to transport sugarsthat are the same size as those transported by wild-type LamB.

Similar results were obtained with sensitivity to bacterio-phage � (Table 2). Susceptibility to � was determined by cross-streaking each strain against �vir. Cells producing wild-typeLamB are sensitive to �. In contrast, cells producing the teth-ered LamB are resistant to �. LamBDY19T-producing cellswere phenotypically identical to those producing wild-typeLamB. These mutants are � sensitive. In contrast,LamBDY6L- and LamBYY6C-producing cells were slightlyresistant to �. The LamBYY6S strain grew poorly on malto-dextrins as a carbon source and was resistant to �, as might beexpected due to the decreased translation previously described(19). The above observations suggested that functional LamBis present, albeit at different levels, in each of the suppressorstrains isolated.

Suppressors of lamBDY and lamBYY produce stable LamBproteins. We initially examined the LamB steady-state proteinlevels by Western blot analysis using antibodies to LamBmonomer. Our results not only gave us insight into the LamBlevels in each strain but also into the molecular weight of themajor species of LamB protein present. Cells carryinglamBDY19T produce a LamB protein that appears equivalentto that from wild-type cells, both at steady-state levels and inmolecular weight (Fig. 2). In contrast, lamBDY6L andlamBYY6C mutants produce steady-state levels of LamB thatare similar to those for wild-type cells, but the molecularweight of the LamB protein is equivalent to that of the teth-ered LamBDY and LamBYY (Fig. 2). The LamB proteinlevels are quite low in the lamBYY6S mutant. Again, this isexpected due to the reduced translation in this mutant. Themolecular weight of LamBYY6S is equal to that of tetheredLamBDY and LamBYY because the signal sequence is notcleaved. Additionally, levels of tethered LamB are low because

the mutant proteins are unfolded in the periplasm and conse-quently degraded by periplasmic proteases.

Suppressors of lamBDY do not restore wild-type signal se-quence processing. Crude outer membranes were preparedfrom each suppressor strain, and protein profiles of these ex-tracts were analyzed by SDS-PAGE and Coomassie staining(Fig. 3A). Cells carrying suppressors of tethered LamB exhibitan induced PspA stress response, as do cells producing teth-ered LamBDY and LamBYY (Fig. 3A). The reasons for thisinduction are not clear, but it appears that the suppressors thatwe have isolated do not entirely relieve the stresses caused bythe cleavage site mutations.

The N-terminal amino acid sequence of LamB protein re-covered from the crude outer membrane preparations wasdetermined (Fig. 3B). As expected, wild-type LamB waspresent in the outer membrane as only the mature protein.LamBDY19T exists in the outer membrane with the last fouramino acids of the signal sequence at its N terminus (Fig. 3B).Clearly, this mutation creates an alternate processing site. Wemight expect such a result because the replacement of a bulkymethionine with a smaller threonine produces a sequence(TSA) very close to the consensus site (AXA) recognized bysignal peptidase (41). Levels of this mutant appeared to bequite low in the outer membrane. Upon further analysis, wedetermined that processing of this mutant is more than fourtimes slower than that of wild-type LamB (data not shown).We hypothesize that slowed processing leads to unfoldedLamB in the periplasm for an extended period of time, result-ing in degradation of a significant fraction of the protein byperiplasmic proteases.

Much more surprising are the results of protein sequencingof the charge change mutant. We found that LamBDY6L thatwas recovered from outer membrane preparations retained the

FIG. 2. Steady-state levels of LamB are increased relative to thoseof tethered mutants in strains carrying suppressors, with the exceptionof LamBYY6S. Strains were grown to mid-log phase in LB medium.Western analysis was done as already described using antibodies toLamB monomer, LamB trimer, and MBP. The LamB trimer antibodyrecognizes OmpA and thus serves as an internal loading control.

TABLE 2. Maltodextrin utilization and � sensitivity phenotypes oflamBDY and lamBYY suppressors

Relevant genotype Dex phenotypea � phenotypeb

lamB� Red SlamBDY White RlamBYY White RlamBDY19T Red SlamBDY6L Pink S/RlamBYY6C Pink S/RlamBYY6S White R

a Dex phenotype was characterized by scoring colony color on dextrin Mac-Conkey agar. Red, Dex�; pink, Dex�/�; White, Dex�.

b � phenotype was analyzed by cross-streaking each strain against �vir. S,sensitive; S/R, slightly resistant; R, resistant.



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entire signal sequence of LamB (Fig. 3B). Despite the pres-ence of this 25-amino-acid extension, LamB remained func-tional and stable in the outer membrane.

A novel LamB species is present in cells carrying lamBDY6L.As discussed in the introduction, LamBDY and LamBYY aretethered to the inner membrane where they are degraded.Several types of suppressors of the toxicity caused by thistethering have been isolated, but they have resulted in non-functional LamB localized either to the inner or outer mem-brane (11). We sought to determine whether the chargechange suppressors of lamBDY isolated in this study couldform stable trimers in the outer membrane despite the pres-ence of the entire signal sequence. To investigate the proper-ties of LamB in each of the suppressor mutants further, weperformed Western blot analysis in which cells were gentlylysed to allow for the recovery of LamB folding intermediates(Fig. 4). Briefly, wild-type cells and those carrying the chargechange mutation were lysed as previously described to preventthe dissociation of higher-molecular-weight intermediates andthe LamB trimer (26). Samples were resuspended in sample

buffer and heated to 37, 55, 70, or 100°C. After SDS-PAGEanalysis, samples were transferred to a polyvinylidene difluo-ride membrane and then exposed to antibodies recognizingLamB monomer, LamB trimer, and MBP (26). As expected,wild-type LamB trimer dissociates completely into unfoldedmonomer above 70°C (Fig. 4). Surprisingly, despite the func-tionality of LamBDY6L, a protein species corresponding towild-type trimer does not exist. Rather, a novel species is ob-served in samples from these charge change mutants (Fig. 4).This species denatures into LamB monomer at temperaturesbetween 37 and 55°C. In addition, we are able to observefolded LamB monomer in the charge change mutant back-ground at 37°C (Fig. 4). This result is intriguing because foldedLamB monomer has not been observed in all strain back-grounds. Rouviere and Gross reported the existence of thefolded monomer intermediate of LamB in strain MC1061 (36).The data presented in Fig. 4 suggest that we may have slowedLamB assembly in the charge change mutants, thus increasingthe levels of LamB folding intermediates.

LamB localizes to the outer membrane in the lamBDY6L

FIG. 3. (A) Crude outer membrane preparations of LamBDY suppressors. LamB and PspA (identified using anti-PspA antibody; data notshown) 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 doneon LamB obtained from the crude outer membranes shown in panel A. Mature LamB protein is indicated by the stippled bar, and the LamB signalsequence is indicated by the white bar. The positions of signal sequence cleavage are indicated by the arrows.



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mutant. To clearly demonstrate the cellular localization of thisspecies, we performed membrane fractionations. The innerand outer membranes were separated by sucrose gradient cen-trifugation, and the resulting fractions were analyzed by Coo-massie staining. The fractions corresponding to inner and

outer membranes were analyzed further by Western analysis(Fig. 5).

In order to detect LamB folding intermediates, which arepresent at low levels, the gel shown in Fig. 5 was purposelyoverexposed. Consequently, proteins such as OmpA that are

FIG. 4. A novel LamB species that denatures at low temperatures is observed in the charge change suppressor. Cells were gently lysed asdescribed in Materials and Methods. Samples were heated to either 37, 55, 70, or 100°C prior to SDS-PAGE. Western analysis was done usingantibody to LamB trimer, LamB monomer, or MBP. The positions of the various LamB species and MBP are noted by the arrows. The LamBtrimer 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

FIG. 5. Folded LamBDY6L fractionates with the outer membrane. Cells were fractionated as described in Materials and Methods. Fractionswere visualized on a Coomassie-stained gel. The fractions corresponding to the inner and outer membranes were subjected to Western blotanalysis. Prior to SDS-PAGE, outer membrane samples were heated to either 37 or 100°C as noted above each lane. LamB folding intermediatesare indicated by the arrows. The LamB trimer antibody recognizes OmpA and thus serves as an internal loading control.



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present at very high levels appear in all fractions. In controlexperiments we showed that the outer membrane proteinOmpF and an inner membrane protein of unknown function ofapproximately 55 kDa (5) fractionate correctly (data notshown).

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

The location of the folded LamB monomer has not beenreported. Our fractionation experiments show that foldedLamB monomer localized to the outer membrane (Fig. 5). Wealso fractionated strain MC1061 for which the folded mono-mer has been clearly demonstrated (36). Consistent with ourobservations above, the folded monomer of LamB localized tothe outer membrane in MC1061, although the levels of thisspecies in a wild-type strain are clearly lower than those ob-served in the charge change mutant (Fig. 5). We are unable todetermine if the folded monomer is actually inserted into theouter membrane or whether it is associated with the membraneat its periplasmic face. Again, we propose that the lamBDY6Lmutant has resulted in slowed LamB assembly that has allowedclear visualization of the folded monomer in fractionation ex-periments.

LamB forms multimers in the lamBYY6C mutant. In orderto understand the identity of the novel species observed withthe lamBDY6L mutant, we took advantage of another suppres-sor that was isolated in the study and found to behave identi-cally to lamBDY6L in phenotypic assays and Western blotanalysis. This mutant, lamBYY6C, substitutes the highlycharged arginine at position six of the signal sequence with anuncharged cysteine residue. We hypothesized that the mutantsthat retain the signal sequence but are functional are posi-tioned as multimers in the outer membrane with their signalsequences clustered together in a bundle such that the LamBpore is not obstructed by them. This being the case, we pro-posed that two of the cysteine mutant signal sequences wouldhave the potential to form a disulfide bond with each other.

To test the possibility that the charge change mutants havethe ability to form multimers, we prepared lysates of thelamBYY6C mutant using the gentle lysis procedure describedabove, with the exception that IAA was added to the lysisbuffer. IAA interacts with free sulfhydryl groups to prevent theformation of disulfide bonds by oxidation during sample han-dling. Thus, disulfide bonds observed must have been formedin vivo. Following cell lysis, we incubated samples at varioustemperatures under both reducing (in the presence of �-mer-captoethanol) and nonreducing (in the presence of IAA) con-ditions, 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 reduc-ing conditions behaved identically to lamBDY6L mutants pre-pared under the same conditions. At the low temperature(37°C), the novel band observed for the lamBDY6L mutant wasalso present for the lamBYY6C mutant and, similarly, this banddenatures to LamB monomer at or below 55°C. In contrast,when the lamBYY6C mutant was prepared and electropho-resed under nonreducing conditions, the novel band was sta-bilized and persisted until temperatures exceeded 70°C (Fig.6). Temperatures at or above 55°C did result in the accumu-lation of some LamB monomer and a species of a highermolecular weight (Fig. 6). At 100°C, the novel species dena-tured completely, and LamB monomer and a species that cor-responds to a molecular mass of approximately 90 kDa ap-peared (Fig. 6). This molecular size is close to that of twoLamB monomers, indicating that under nonreducing condi-tions, LamBYY6C is able to form a disulfide-bonded dimer.We also note that a small amount of LamB dimer was presentat 37°C. We believe that this is due to inadvertent heating thatmay have occurred during lysis (specifically centrifugation) ofthe samples. At 100°C, an additional protein with a slightlyfaster mobility than the LamB dimer was present (Fig. 6). Wesuggest that in addition to the intermolecular disulfide bondthat forms in the lamBYY6C mutant, this species also containsan intramolecular disulfide bond involving one or both of thecysteines that are located in one of the surface loops of LamB.

FIG. 6. LamB charge change mutants from multimers. Cells carrying lamBYY6C were gently lysed and treated with either �-mercaptoethanolor 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



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Another possibility is that the lower-molecular-weight proteinis LamB monomer that is also disulfide bonded to anotherprotein. Intramolecular disulfide bonding between the two cys-teines on surface loop 1 of LamB has been reported (22, 23).We have not yet investigated these possibilities further. TheLamB monomers that accumulate at temperatures above 37°Ccould be components of the trimer that were not participantsin disulfide bonding. Therefore, these experiments suggest thatLamB mutants that retain their entire signal sequence are, infact, able to form multimers.

lamBDY6L slows the formation of LamB folding intermedi-ates. We hypothesized that in the charge change mutant, as-sembly of LamB might be slowed due to the presence of theentire signal sequence. To test this idea, we followed the ki-netics of LamB trimerization in both wild-type cells and thelamBDY6L mutant. Figure 7 shows a pulse-chase experimentcalled a trimer assay (26). Cells were labeled with [35S]methi-onine followed by a native immunoprecipitation with antibod-ies that recognize the monomer form of LamB, the trimer, andMBP as a loading control (26). Following immunoprecipita-tion, samples were resuspended in sample buffer and heated toeither 37 or 70°C. In wild-type cells, heating to 70°C strippedthe lipopolysaccharide (LPS) from the trimer and denaturedthe LamB assembly intermediates. SDS-PAGE analysis al-lowed the separation of the trimer from the denatured mono-mer LamB. At low temperatures, wild-type LamB trimerschased to LPS-associated trimer by approximately 2 postchase(Fig. 7, top). At 70°C, the trimers were dissociated from LPSand thus had a faster mobility (Fig. 7, bottom).

We noticed several differences in the formation of LamBfolding intermediates with the lamBDY6L mutant. First, thetransition from LamB monomer to a higher-molecular-weightspecies was slowed—monomer was present as late as 10 minpostchase (Fig. 7, top). As mentioned above, in certain strainbackgrounds a folded monomer species has been visualized ina trimer assay (36). In the MC4100 background we are workingwith, the presence of folded LamB monomer is often notobserved. However, we were able to visualize the folded mono-mer species of LamB in the lamBDY6L mutant background(Fig. 7, top). The trimer assay results show that, as with thetransition of unfolded monomer into a higher-molecular-weight species, the disappearance of folded monomer wasslowed in the charge change mutant (Fig. 7, top). Consistentwith previous data, the folded monomer in our experimentsmelted at 70°C (Fig. 7, bottom).

The results of the kinetic experiment in Fig. 7 demonstratethat the novel LamB species observed in Western blot analysisof 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 changemutant background and the kinetics of appearance of thisnovel species suggest that it is a LamB trimer.

DISCUSSION

We report here the isolation of suppressors of tetheredlamBDY and lamBYY. The goal of our work has been to under-stand the targeting and folding of a class of outer membraneproteins collectively termed the �-barrels. As a model, we have

been studying the maltoporin LamB. Efforts to isolate target-ing and folding factors genetically have been generally unsuc-cessful, possibly due to functional redundancy within theperiplasm of E. coli. To exaggerate the defect caused by nullmutations in proteins of redundant function, attempts weremade to isolate lamB mutants slowed in targeting and/or fold-ing. In particular, mutations that hinder cleavage of the LamBsignal sequence by signal peptidase were constructed. Thesemutants resulted in leaky phenotypes which eventually tar-geted mutant LamB (6) or in LamB molecules which werecompletely blocked in signal sequence processing, resulting inthe tethering of the protein to the inner membrane by its signalsequence (10). The former LamB mutants are targeted asefficiently as wild-type LamB (6), and the latter mutationsresulted in LamB protein that was no longer functional be-cause it was tethered to the inner membrane (10).

In this study, we have isolated functional suppressors oflamBDY and lamBYY. The two classes of suppressors are dis-tinct. We have shown that one mechanism of lamBDY suppres-sion is the creation of a novel signal sequence cleavage site.Another, distinct mechanism is the relief of a strong positivecharge at the amino terminus of the signal sequence. Interest-ingly, others have predicted that inner membrane proteins arepositioned in the membrane such that positively charged res-idues located in the cytoplasmic portion of the signal sequenceserve to anchor transmembrane domains (4). The chargechange mutants that we have isolated offer further support forthis “positive inside rule.” The arginine that is found in thewild-type LamB signal sequence serves as an anchor that re-tains the signal sequence in the inner membrane. When thisarginine is changed to an uncharged amino acid, such asleucine or cysteine, the strength of the anchor is reduced,allowing the signal sequence to slip through the inner mem-brane. Once LamB is released from the inner membrane, it canlocalize to the outer membrane as a multimer.

The lack of signal sequence cleavage in the charge changemutant is not without consequences. We have shown that thecharge change mutants are slowed in assembly. This may resultfrom the mechanism that mutant LamB utilizes to escape fromthe inner membrane. It is tempting to speculate that this mech-anism involves periplasmic folding factors that would normallybe involved in the proper folding of wild-type LamB. In thiscase, the partial folding of LamB catalyzed by these factorsmay provide the energy necessary to pull the signal sequencethrough the inner membrane.

Regardless of the mechanism by which the charge changeLamB mutants are released from the inner membrane, theslowed assembly has allowed us to follow the folding of LamBand to visualize folding intermediates that are not seen in cellscarrying wild-type LamB. In certain strain backgrounds, theexistence of a folded monomer intermediate of LamB has beenobserved (36). We have not been able to observe the foldedmonomer in the MC4100 wild-type background. However, inthe charge change mutants, slow assembly reveals a form ofLamB that, like the folded monomer previously reported,melts into a higher-molecular-weight form of LamB. In thecase of the charge change mutant, the folded monomer meltsinto a precursor LamB owing to the presence of the signalsequence.

Although the existence of the folded monomer has been



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shown using biochemical approaches (36), the cellular local-ization of this folding intermediate has not been elucidated.We have shown that the folded monomer localizes to the outermembrane. We cannot determine whether the folded mono-mer is positioned within the outer membrane or whether it isassociated in some other fashion. Nonetheless, this is an in-

triguing result because it suggests that the LamB trimers as-semble in the outer membrane. The idea that LamB trimer-ization occurs at the outer membrane rather than in theperiplasm has been proposed in previous studies (26).

It has proven difficult to determine whether the novel-mo-lecular-weight species observed in the charge change mutants

FIG. 7. A novel species recognized by LamB trimer antibody is present in the charge change mutants. Cells carrying either wild-type lamB orlamBR6LA23DA23Y were subjected to pulse-chase labeling followed by native immunoprecipitation with antibodies recognizing unfolded LamBmonomer, 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 MBPare noted by the arrows. p, precursor; m, mature.



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is actually LamB trimer. It is impossible to predict how a LamBtrimer with three additional hydrophobic sequences (the signalsequences) will run in SDS-PAGE. Because strains carryingthese mutations are LamB�, we favor the proposal that thenovel band is a LamB trimer that, due to its increased hydro-phobicity, binds more SDS and thus runs faster through the gelthan wild-type LamB trimer. We do know that LamB forms amultimer of at least two monomers in the outer membrane.Proof for this lies in the fact that when run under nonreducingconditions, the novel band is stabilized in the cysteine chargechange mutant. In addition, this species melts into a proteinthat has a molecular weight equal to two LamB monomers.The fact that we also observe a protein that has a molecularweight equal to that of the LamB monomer in these prepara-tions may provide further evidence for trimer formation. Ob-viously, only two LamB monomers can participate in dimerformation by disulfide bonding.

An alternative explanation for the novel species is that it isa LamB dimer. There has been little evidence for the existenceof �-barrel dimers even though dimers must be an obligatoryintermediate in trimer formation. Two studies have suggestedthat OmpF and OmpC form dimers that are loosely associatedwith the outer membrane (33, 35). However, it seems unlikelythat a �-barrel dimer would be functional in the outer mem-brane. This would require proteins like LamB to have verylarge hydrophilic surfaces contacting the very hydrophobic en-vironment of the membrane, and the stability of such a proteinin the outer membrane is questionable.

We favor the possibility that LamB can form stable trimerseven with the signal sequence attached. Interestingly, a prece-dent for such trimers does exist. The sucrose-specific porin(ScrY) of Salmonella enterica serovar Typhimurium has beenshown to have high structural homology to LamB (18). Themajor difference between ScrY and LamB lies in a periplasmic70-amino-acid extension at the N terminus of the ScrY protein(14). Both LamB and ScrY possess aromatic residues withinthe pore of the �-barrel that have been postulated to form aslide through which sugars could pass en route to the periplasm(14, 38). The N-terminal extension of ScrY is thought to be anextension of this sugar slide (14). We have shown that a LamBmutant retaining its signal sequence is functional, but not asfunctional as the wild-type LamB porin. Therefore, it wouldseem that an N-terminal extension such as that seen in ScrY isnot necessarily desirable in other bacterial porins, such asLamB.

In this study, we have isolated a functional suppressor oftethered LamB. As mentioned above, lamBDY was origi-nally constructed to slow down the assembly of LamB in theouter membrane. Instead, however, these mutations re-sulted in a LamB protein that is not released from the innermembrane. We have shown that the charge change mutantrelieves the tethering and allows assembly, albeit slowed, inthe outer membrane. The mechanism by which LamBDY6Land LamBYY6C are released from the inner membrane isintriguing. It is possible that these mutants leave the innermembrane spontaneously. Alternatively, the LamB signalsequence may be pulled out of the membrane by periplasmicfolding factors.

ACKNOWLEDGMENTS

We thank members of the Silhavy laboratory for helpful discussionsand for critically reading the manuscript. We are grateful to SusanDiRenzo for her help in preparing the manuscript.

A.R.D. was supported by an NIH departmental training grant (GM07388); T.J.S. was supported by an NIGMS MERIT grant (GM34821).

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