Protease-Deficient DegP Suppresses Lethal Effects of a Mutant OmpC Protein by Its Capture

The expression of assembly-defective outer membrane proteinscan confer lethality if they are not degraded by envelope proteases.We report here that the expression of a mutant OmpC protein,OmpC_2Cys, which forms disulfide bonds in the periplasm due tothe presence of two non-native cysteine residues, is lethalin cells lacking the major periplasmic protease, DegP. Thislethality is not observed in dsbA strains that have diminishedability to form periplasmic disulfide bonds. Our data show thatthis OmpC_2Cys-mediated lethality in a degP::Km^r dsbA⁺ backgroundcan be reversed by a DegP variant, DegP_S210A, that is devoidof its proteolytic activity but retains its reported chaperoneactivity. However, DegP_S210A does not reverse the lethal effectof OmpC_2Cys by correcting its assembly but rather by capturingmisfolded mutant OmpC polypeptides and thus removing them fromthe assembly pathway. Displacement of OmpC_2Cys by DegP_S210Aalso alleviates the negative effect that the mutant OmpC proteinhas on wild-type OmpF.

INTRODUCTION

Top

Introduction

OmpC and OmpF represent a major class of Escherichia coli outermembrane proteins (OMPs). These proteins form water-filled channelsthat permit the diffusion of small solutes into the cell. Becauseof this property, OmpC and OmpF are called porins or sometimes"classical" porins, which refer to their stable ß-barrelstructure and nonspecific solute diffusion properties (17).Each porin monomer consists of a 16-stranded antiparallel ß-barrel(22). These porins exist as homotrimers, in which three barrelsassemble to produce three separate channels (22).

Besides their atomic structures, the assembly and targetingof the outer membrane ß-barrel proteins have beenan intense topic of study (for a recent review, please see reference6). Based on studies conducted in several laboratories, it hasbeen shown that the assembly of porin proteins begins with theproteolytic cleavage of an amino-terminal signal sequence thatyields soluble nascent monomers. Rapid folding of these monomersis followed by their oligomerization into protease- and heat-sensitivetrimers called metastable trimers, which slowly mature intoprotease- and heat-resistant trimers, the final product of assembly.

Although porins in vitro can be fully folded into their nativetrimeric structures from denatured monomers without the needof an accessory protein (7, 27), assembly in vivo is highlydependent on several cellular folding factors. Among them arespecific periplasmic foldases such as SurA and FkpA, which catalyzecis-trans peptidyl-prolyl isomerization (16, 20), although ithas never been demonstrated that this enzymatic activity isactually involved in OMP assembly. Consistent with this notion,Behrens et al. (3) have recently shown that SurA assists inOMP assembly independent of its isomerase activity. Anotherperiplasmic foldase, DsbA, is involved in promoting correctdisulfide bond formation of certain OMPs (2). The wild-typesequence of the classical porins, however, lacks cysteine residuesand thus they assemble independently of DsbA's activity. Incontrast to wild-type OmpC, the assembly of a mutant OmpC protein,OmpC_2Cys, containing two non-native cysteine residues (C74 andC154) is drastically influenced by DsbA (11). Since OmpC_2Cysis the subject of the present study, it is further discussedbelow.

Another periplasmic protein, Skp, is thought to assist in OMPassembly by acting as a general chaperone (5, 21). However,the skp-null mutant phenotype showing a dramatic reduction inOMP levels could not be repeated in other laboratories sinceit was first reported. Recent genetic studies have shown thatthe presence of skp- and surA-null alleles in a strain confersa synthetic lethal phenotype (19). Based on these studies, ithas been proposed that Skp and SurA are components of separatefolding pathways providing chaperone activities (19). Nonproteinaceouscomponents, including phospholipids and lipopolysaccharide (whichis exclusively localized on the outer leaflet of the outer membrane),are also important for OMP assembly (1, 9, 18).

Periplasmic and envelope proteases become particularly importantwhen the machinery that ensures correct OMP assembly malfunctionsor the primary sequence of an OMP endures an unacceptable alteration.One periplasmic protease, DegP, has received the most attentionin connection with OMP assembly. degP-null mutants do not growat temperatures above 39°C (10, 25, 26), presumably becauseof a greater need for the removal of misfolded proteins at elevatedtemperatures. However, expression of certain misfolded OMPsrenders the DegP's presence essential even at lower growth temperatures(14). Elegant work carried out by Spiess et al. (25) showedthat DegP, in addition to proteolysis, assists in the correctfolding of a periplasmic protein. A recent genetic study alsoargued for DegP's involvement in providing periplasmic chaperoneactivities (19); however, this contention was not backed bydirect biochemical evidence.

We have previously described the isolation of a novel classof OmpC mutants that affected either the channel or phage receptorproperty of the protein (13, 28). One such OmpC mutant carryinga R74C substitution functionally enlarged the channel to permitthe entry of large sugars such as maltodextrins, which are normallyexcluded from the OmpC channel (13). This isolate was subsequentlyused for the isolation of phage-resistant mutants under conditionsthat demanded the retention of a functional OmpC channel butno phage receptor activity. A group of OmpC mutants from thisselection contained a G154C substitution while retaining theoriginal R74C alteration (11). This mutant OmpC protein, OmpC_2Cys,which now had two non-native cysteine residues in its maturesequence, conferred phage resistance due to a biogenesis defect(11).

The OmpC_2Cys biogenesis defect was shown to stem from the protein'sability to form disulfide bonds in DsbA⁺ cells (30); OmpC_2Cysassembles normally in a genetic background lacking DsbA. OmpC_2Cysproduces a novel transdominant effect on OmpF (11). Since theeffect on OmpF is specific, this suggests that either the inhibitionwas due to the mixing of mutant OmpC assembly intermediateswith that of OmpF or that the two proteins share a common assemblyfactor.

Just as noted for several assembly-defective OmpF mutants (14),it was discovered that the expression of OmpC_2Cys in a DsbA⁺background obligated the presence of DegP at all growth temperatures.As anticipated, in a background lacking DsbA, and hence witha diminished disulfide forming ability in the periplasm, theabsence of DegP produced no lethal effects at growth temperaturesof $<=$ 37°C.

We investigated here the effect of a protease-deficient DegP,DegP_S210A, on OmpC_2Cys-mediated lethality in a degP-null strain.Our findings provide direct biochemical evidence showing thatDegP_S210A alleviates OmpC_2Cys's lethal effects not by improvingits folding or assembly but rather by sequestering misfoldedmutant OmpC polypeptides and thus removing them from the normalassembly pathway.

MATERIALS AND METHODS

Top

Materials and Methods

Media, reagents, and bacterial strains. Luria broth medium, Luria agar (1.5% [wt/vol]) medium, and M63salt-based minimal medium were prepared according to the methodof Silhavy et al. (23). When cultures were grown on minimalmedium, 0.4% glycerol and 0.1 mM amino acid mixture lackingmethionine were added. When required, ampicillin (50 µg/ml),kanamycin (25 µg/ml), chloramphenicol (25 µg/ml),tetracycline (25 µg/ml), and IPTG (isopropyl-ß-D-thiogalactopyranoside;0.4 mM) were added to the growth medium. Heat-killed Staphylococcusaureus (Pansorbin) was purchased from Calbiochem. [³⁵S]methionineExpre protein labeling mixture was purchased from Du Pont-NewEngland Nuclear. All other chemicals were of analytical grade.The bacterial strains used in the present study are listed inTable 1.

View this table:
[in this window]
[in a new window]
TABLE 1. Strains used in this study

Pulse-chase labeling. Pulse-chase labeling was performed essentially as describedpreviously (14). Cells were grown overnight at 37°C in glycerolminimal medium supplemented with 0.1 mM amino acid mixture.After a 1-in-30 dilution of overnight culture into fresh mediumsupplemented with 0.4 mM IPTG, cells were grown to an opticaldensity at 600 nm of 0.3. Cells were pulse-labeled with [³⁵S]methionine(20 µCi/ml) for 20 s and then chased with cold methionine(20 mM) in the presence of a protein synthesis inhibitor, spectinomycin(200 µg/ml). Samples (0.5 ml) were taken 0.5, 1, 2, 5,10, and 20 min postchase and immediately mixed with 0.5 ml ofice-cold 15% trichloroacetic acid. Cells were centrifuged at12,000 x g for 5 min at 4°C. Pellets were washed once with0.5 ml of 5% trichloroacetic acid and once with 95% ethanoland then dried under a vacuum. Dried pellets were resuspendedin 200 µl of sodium dodecyl sulfate (SDS) buffer (20 mMTris-HCl, pH 7.5; 10 mM EDTA; 1% SDS) and boiled for 5 min.Cell lysates (100 µl) were transferred into tubes containing1 ml of the immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5;20 mM NaCl; 1% Triton X-100) and MalE and LamB antibodies. Tubeswere incubated overnight at 4°C on a rocker. To bring downimmunocomplexes, heat-killed S. aureus cells (50 µl) wereadded, and tubes were incubated at 4°C on a rocker for atleast 4 h. Immunocomplexes were recovered by centrifugationand washed twice in immunoprecipitation buffer, twice in Trisbuffer (10 mM Tris-HCl; pH 7.5), and once in water. Washed pelletswere resuspended in sample buffer, boiled, and analyzed by SDS-polyacrylamidegel electrophoresis (PAGE). Gels were fluorographed, dried,and exposed to X-ray films at -85°C. Radioactive proteinbands were quantified by using a Bio-Rad gel analyzer.

Membrane analysis. Whole-cell envelopes were extracted by the French cell lysismethod as described previously (12). The detergent solubilityof membrane proteins was tested by mixing an equal amount ofmembrane and 2% Sarkosyl in 20 mM Tris-HCl (pH 7.5). The mixturewas incubated for 30 min at room temperature and then centrifugedin a Beckman benchtop ultracentrifuge at 105,000 x g for 30min at 4°C with a TLA 120 rotor. Both the supernatant andthe detergent-insoluble pellet were recovered for protein analysis.

Separation of inner and outer membranes from whole-cell envelopeswas achieved by sucrose (30 to 55%) density gradients accordingto the method of Misra et al. (14). After centrifugation, fractionswere collected and the refractive index for each fraction wasdetermined at 25°C. Densities for each fraction were calculatedby their respective refractive index. Samples from each fractionwere analyzed by SDS-PAGE.

Interaction of OmpC_2Cys with DegP_S210A. In vivo interactions between OmpC_2Cys and DegP_S210A were examinedby purifying the soluble His tag variant of DegP_S210A (DegP_S210A-6His)through Ni²⁺ affinity chromatography. Subsequently, fractionscontaining DegP_S210A-6His were examined for the presence ofOmpC_2Cys by Western blot analysis. Briefly, 5 ml of cells containingpCS21 (in which the expression of DegP_S210A-6His is inducibleby IPTG) were grown to an optical density at 600 nm of 0.2 inthe presence of 0.4 mM IPTG. Cells were pelleted and resuspendedin 2 ml of 10 mM imidazole containing 20 µl of freshlyprepared lysozyme (100 mg/ml). Cells were lysed by a small Frenchpressure cell and, after the removal of unlysed cells by low-speedcentrifugation, lysates were centrifuged at 105,000 x g in a50 Ti rotor for 1 h at 4°C to pellet the membranes. DegP_S210A-6Hiswas purified from the supernatant by affinity chromatographyin a Ni²⁺-nitrilotriacetic acid column according to the PharmaciaBiotech HisTrap purification protocol, except that the elutionof DegP_S210A-6His was carried out in several steps involvinga gradual increase in the imidazole concentration from 20 to200 mM. The DegP_S210A-6His protein eluted with 200 mM imidazole.

Proteins were analyzed by SDS-PAGE and transferred into ImmunoLite membranes by using a Mini Transblot electrophoretic transfercell (Bio-Rad). Blotted proteins were incubated for 90 min withprimary antibodies raised against DegP (1:10,000 dilution) andOmpC (1:10,000 dilution) and then with the secondary antibody(goat anti-rabbit immunoglobulin G) for 1 h. Detection was carriedout according to the manufacturer's protocol (Bio-Rad). Anti-DegPantibodies were kindly provided by Jon Beckwith (Harvard).

Protein density levels from Coomassie blue-stained gels andWestern blots were determined by using Scion Image Analysissoftware.

RESULTS AND DISCUSSION

Top

Results and Discussion

Overexpression of a protease-deficient DegP_S210A eliminates the toxic effect of OmpC_2Cys. We have previously observed that the expression of assembly-defectiveOmpF mutants is lethal in a DegP^- background (14). This is presumablybecause DegP degrades misfolded proteins, thus eliminating theirtoxic effects. As found for the assembly-defective OmpF mutants,the expression of OmpC_2Cys also demanded the presence of DegPfor cell viability. Initially, this conclusion was reached dueto our inability to introduce, through P1 transduction, theompC402 (OmpC_2Cys) allele into a degP::Km^r strain. Subsequently,direct evidence for this synthetic lethality was obtained bydemonstrating that the ompC402 allele could be transduced intoa degP::Km^r background only when the expression of a protease-deficientDegP_S210A protein was simultaneously induced from a plasmidreplicon (Fig. 1). In contrast, the parental ompC234 (OmpC_1Cys)allele was transducible into a degP::Km^r background at the expectedfrequency independent of DegP_S210A's expression (Fig. 1B).

View larger version (47K):
[in this window]
[in a new window]
FIG. 1. (A) Diagram showing two possible P1-mediated crossover events between the donor (ompC234 [OmpC_1Cys] zei::Tn10 or (ompC402 [OmpC_2Cys] zei::Tn10) and the recipient (ompC'::lacZ⁺ degP::Km^r/pdegP_S210A) DNA. The cotransductional linkage between the zei::Tn10 (the selectable marker) and ompC is 50%. A crossover between 1 and 2 will yield tetracycline-resistant (Tc^r) Lac⁺ (ompC'::lacZ⁺) transductants, whereas a crossover between 1 and 3 will yield Tc^r Lac^- (ompC) transductants. (B) P1 transduction results drawn from diagram presented in panel A.

DegP_S210A does not stimulate OmpC_2Cys degradation. Since substitution of the only serine residue of DegP to alanine(S210A) is known to make it proteolytically inactive (29), itis unlikely that the rescue from OmpC_2Cys-mediated lethalityby DegP_S210A involved degradation of the mutant protein. Tosubstantiate this, we first confirmed that the overexpressionof DegP_S210A did not directly or indirectly induce proteolysisof mutant envelope proteins. For this we utilized a LamB amberfragment whose in vivo degradation is largely dependent on DegP(Fig. 2). Pulse-chase experiments showed that overexpressionof DegP_S210A did not increase the rate of degradation of theLamB amber fragment (Fig. 2). Instead, overexpression of DegP_S210Ain a DegP⁺ background increased the half-life of the LamB amberfragment (Fig. 2), thus suggesting a protective rather thana destructive role for the variant DegP_S210A protein. This protectiverole of DegP_S210A was most apparent in a background devoid ofwild-type DegP. These results clearly demonstrated that theoverexpression of DegP_S210A did not involve increased proteolyticactivity in vivo.

View larger version (24K):
[in this window]
[in a new window]
FIG. 2. In vivo degradation of a LamB amber fragment in various genetic backgrounds. Freshly grown cells were subjected to pulse-chase analysis as described in Materials and Methods. LamB and MalE (as a gel loading control) were immunoprecipitated from [³⁵S]methionine-labeled protein extracts obtained from DegP⁺ ( ${square}$ ), DegP^- ( ${blacksquare}$ ), DegP⁺/DegP_S210A ( ${circ}$ ), and DegP^-/DegP_S210A (•). The highest LamB level was taken as 100, and the other LamB values were adjusted accordingly.

Next, we tested the effect of DegP_S210A overexpression on mutantOmpC and wild-type OmpF. As found for the LamB amber fragment,the level of OmpC_2Cys in the envelope increased when DegP_S210Awas overexpressed (Fig. 3). This result confirmed that the rescuefrom lethality did not involve degradation of the mutant protein.As mentioned earlier, the expression of OmpC_2Cys imposes a negativeeffect on the assembly of the wild-type OmpF protein, presumablyby interfering with the oligomerization of OmpF monomers orsequestering a common assembly factor (11). Under conditionsin which DegP_S210A overexpression elevated OmpC_2Cys levels,it also elevated OmpF levels, thus abolishing the negative effectof OmpC_2Cys on OmpF (Fig. 3, compare lanes 2 and 4).

View larger version (44K):
[in this window]
[in a new window]
FIG. 3. Effect of DegP_S210A on OmpC and OmpF. (A) Envelopes obtained from DegP⁺ OmpC_1Cys (lane 1), DegP⁺ OmpC_2Cys (lane 2), DegP_S210A OmpC_1Cys (lane 3), and DegP_S210A OmpC_2Cys (lane 4) were examined by SDS-PAGE. OmpC, OmpF, and OmpA bands were identified by Western blot analysis. (B) OmpC (solid bars) and OmpF (open bars) levels from panel A were quantified relative to OmpA.

DegP_S210A increases the level of improperly assembled OmpC. The elevation of OmpC_2Cys levels and a concomitant increasein wild-type OmpF levels suggested that overexpression of DegP_S210improved the envelope's assembly or folding environment. Consistentwith this notion, it was demonstrated by Spiess et al. (25)that the protease-deficient DegP_S210A protein promoted properfolding of a periplasmic protein MalS. Also, based on geneticdata, it has been recently argued that DegP may be involvedas a general chaperone in the folding and assembly of OMPs (19).We therefore tested the possibility that the elevated OmpC_2Cyslevels caused by DegP_S210A overexpression may be due to itsimproved assembly. For this we examined the solubility of membrane-boundOmpC in the anionic detergent Sarkosyl, which is known to solubilizeinner membrane proteins and proteins that are loosely boundto the outer membrane. Since improperly assembled OMPs may notbe correctly inserted in the outer membrane, they could be solubilizedby Sarkosyl.

In the absence of DegP_S210A overexpression, the parental andmutant OmpC proteins fractionated with the membrane pellet fromuntreated envelopes (Fig. 4, lanes 1 and 2). However, when DegP_S210Awas overexpressed, a significant portion of OmpC_2Cys, and tosome extent OmpC_1Cys, became soluble even without detergenttreatment (Fig. 4, lanes 3 and 4). When membranes obtained fromcells not overexpressing DegP_S210A were treated with Sarkosyl,OmpC_2Cys, like the parental OmpC_1Cys protein, was present primarilyin the Sarkosyl-insoluble membrane pellet (Fig. 4, lanes 5 and6 [top panel]), suggesting that at least as far as detergentsolubility is concerned it was properly inserted in the outermembrane. Detergent treatment of membranes obtained from DegP_S210A-overexpressingstrains dislodged a large quantity of both OmpC species fromthe envelope (Fig. 4, lanes 7 and 8 [bottom panel]). These resultsshowed that the DegP_S210A-mediated increase in OmpC levels didnot lead to the proper assembly or insertion of OmpC in theenvelope. This finding was further supported by the observationthat the increase in the OmpC_2Cys level did not make cells sensitiveto OmpC-specific bacteriophages, reflecting the continued presenceof a conformationally defective OmpC_2Cys. The observation thatoverexpression of DegP_S210A causes the parental OmpC_1Cys proteinto become detergent soluble suggests that porin assembly intermediatesin general can be targeted by DegP_S210A. Consistent with thisnotion we found that a fraction of the assembly-competent wild-typeOmpC protein was also subjected to DegP_S210A-mediated detergentsolubilization (data not shown). The lack of this effect onOmpA by DegP_S210A overexpression may reflect distinct foldingproperties of its assembly intermediates compared to that ofthe trimeric porin proteins.

View larger version (38K):
[in this window]
[in a new window]
FIG. 4. Sarkosyl solubility of membrane-associated OmpC. Purified whole-cell envelopes were treated either with just buffer (Untreated) or with Sarkosyl as indicated. After incubation, samples were centrifuged to separate Sarkosyl-soluble proteins (S, supernatant) from envelopes containing Sarkosyl-insoluble proteins (P, pellet). OmpC from each fraction was detected by Western blot analysis with polyclonal antibodies specific to OmpC. Lanes: 1 and 5, DegP⁺ OmpC_1Cys; 2 and 6, DegP⁺ OmpC_2Cys; 3 and 7, DegP_S210A OmpC_1Cys; 4 and 8, DegP_S210A OmpC_2Cys.

DegP_S210A alters OmpC localization. Since an increase in the mutant OmpC level by DegP_S210A overexpressiondid not lead to its proper assembly or insertion, we were curiousas to its localization in the cell envelope. This was examinedby fractionating whole-cell envelopes into inner and outer membranesby using sucrose density gradients (Fig. 5). OmpC and OmpA (asa control protein) levels were quantified from the inner (buoyantdensities of 1.15 to 1.16) and outer membrane (buoyant densitiesof 1.21 to 1.22) fractions (Table 2).

View larger version (53K):
[in this window]
[in a new window]
FIG. 5. Sucrose density gradient analyses of whole-cell envelopes. Envelopes obtained from DegP⁺ OmpC_1Cys, DegP_S210A OmpC_1Cys, DegP⁺ OmpC_2Cys, and DegP_S210A OmpC_2Cys strains (panels 1 to 4, respectively) were fractionated into inner and outer membranes by sucrose density gradients. After centrifugation, fractions were collected from top and analyzed by SDS-PAGE. Protein bands were visualized by Coomassie blue staining. Fractions corresponding to inner membrane (IM), intermediate density material (M), and outer membrane (OM) are shown on top. Positions of OmpC, OmpA, DegP_S210A, and a protein band ( ${circ}$ ) that primarily localizes with the intermediate-density material are indicated.

View this table:
[in this window]
[in a new window]
TABLE 2. Distribution of OmpC and OmpA in outer and inner membrane fractions

Remarkably, overexpression of DegP_S210A caused the localizationof some OmpC_2Cys molecules to the inner membrane (Fig. 5) suchthat approximately four times more OmpC_2Cys was present therecompared to that in a DegP⁺ background (Table 2). This increasein the inner membrane was not due to an overall increase inmutant protein levels because, in contrast to the inner membrane,the level of OmpC_2Cys decreased in the outer membrane (Table2). Interestingly, the parental OmpC_1Cys protein was also subjectedto this effect of DegP_S210A overexpression (Fig. 5), althougha greater influence was observed for OmpC_2Cys (Table 2). Unlikethese variant OmpC proteins, overexpression of DegP_S210A hadno effect on OmpA's localization (Fig. 5 and Table 2); thus,this DegP_S210A-mediated relocalization was specific to the mutantproteins.

Interestingly, fractionation studies revealed that a significantportion of DegP_S210A was also localized in the inner membrane(Fig. 5). The inner membrane localization of DegP_S210A is consistentwith a detailed fractionation analysis carried out previouslyby Skorko-Glonek et al. (24). The colocalization of DegP_S210Aand OmpC in the inner membrane raises the possibility of directinteractions between these proteins. It is important to mentionhere that our fractionation studies showed that significantamounts of both DegP_S210A and mutant OmpC proteins were alsopresent in the soluble fraction; the data showing enhanced solubilityof mutant OmpC due to increased DegP_S210A expression are presentedin Fig. 4. Experiments to examine a possible direct interactionbetween DegP_S210A and OmpC_2Cys from soluble fractions are describedbelow.

Inner membrane-localized OmpC molecules have characteristics of unassembled polypeptides. Properly assembled porin molecules are resistant to heat denaturationand detergent solubility. Heat sensitivity assays (Fig. 6) showedthat the inner membrane-localized OmpC_1Cys and OmpC_2Cys denaturedinto monomers even when analyzed at room temperature (25°C).On the other hand, outer membrane-localized OmpC proteins displayedheat resistance that is characteristic of properly folded stabletrimers (Fig. 6). Additionally, the inner membrane-localizedOmpC molecules, in contrast to those localized in the outermembrane, could be completely solubilized by Sarkosyl (datanot shown). Together, these results indicated that the innermembrane-localized OmpC molecules most likely represent unassembledpolypeptides.

View larger version (31K):
[in this window]
[in a new window]
FIG. 6. Heat sensitivity of OmpC_1Cys and OmpC_2Cys. Equal amounts of samples from the inner membrane (IM) and outer membrane (OM) were incubated at 25 or 100°C as indicated for 15 min prior to SDS-PAGE. OmpC was detected by Western blot analysis with a mixture of antibodies specific to OmpC and OmpA. OmpA* is a heat-modifiable form of OmpA.

DegP_S210A directly interacts with OmpC_2Cys. As stated above, colocalization of subpopulations of OmpC_2Cysand a fraction of DegP_S210A in both the inner membrane and solublefractions raised the possibility that these two proteins engagein direct interactions. To examine this, a variant DegP_S210Acontaining a His₆ tag was utilized. The His₆-tagged DegP_S210Aprotein (DegP_S210A-6His) was expressed in a background alsoexpressing OmpC_2cys, and the soluble tagged DegP protein waspurified through affinity chromatography with Ni²⁺ columns.Affinity-bound DegP_S210A-6His was eluted with imidazole, andthe eluted material was tested for the presence of mutant OmpC.

Four equal aliquots of the fraction containing eluted DegP_S210A-6Hiswere analyzed in separate lanes on a polyacrylamide gel and,after electroblotting, the filter was cut into strips correspondingto individual lanes and then hybridized with antibodies raisedagainst DegP, OmpC, DegP plus OmpC, or OmpC plus OmpA (Fig.7). The results showed unequivocally that the mutant OmpC_2Cyspolypeptides coeluted with the DegP_S210A-6His protein (Fig.7, lane 2). The absence of OmpA from the eluted DegP_S210A signifiedthe specificity of the DegP_S210A-OmpC interaction (Fig. 7, lane4). These results are consistent with the fractionation analysis(Table 2), which also showed a specific effect of DegP_S210Aon OmpC_2Cys but not on OmpA.

View larger version (29K):
[in this window]
[in a new window]
FIG. 7. Direct interaction between DegP_S210A-6His and OmpC_2Cys. Freshly grown cells expressing DegP_S210A-6His and OmpC_2Cys were lysed as described in Materials and Methods. Membrane-free cell lysates were subjected to Ni²⁺-nitrilotriacetic acid column chromatography to purify DegP_S210A-6His. Four identical samples (lanes 1 to 4) of the eluted fraction containing DegP_S210A-6His were subjected to SDS-PAGE and Western blot analyses with the indicated antibodies. An aliquot of outer membrane containing OmpC and OmpA proteins was analyzed in lane 5.

These results corroborated the notion that overexpression ofDegP_S210A relieved the toxicity of the mutant OmpC protein notby correcting assembly but rather by sequestering it. Capturingof the mutant OmpC_2Cys protein by DegP_S210A also alleviatedthe transdominant effect of mutant OmpC on OmpF's assembly (Fig.3).

DegP and OMP assembly. Both genetic (14, 15, 19) and biochemical (25) studies supportthe notion that DegP plays an important role in the assemblyof envelope proteins. Initially, this was thought to be solelydue to the degradation of mutant envelope proteins. However,the finding of Spiess et al. (25) suggested that DegP is alsoinvolved in the biogenesis of at least one periplasmic protein,MalS, as a general chaperone. Despite this exciting discovery,no other E. coli protein has been shown to be dependent on DegP'schaperone activity for its assembly; thus, the notion that DegPcan act as a "general" chaperone still awaits further biochemicalverification.

The data presented here and published earlier (14) showed thata protease-deficient DegP_S210A, which retains its chaperoneactivity (25), stabilizes assembly-defective OMPs without actuallyimproving their assembly. As demonstrated for OmpC_2Cys in thepresent study, the stabilization of mutant proteins likely involvestheir sequestration by the polypeptide-binding cavity of DegP_S210A(8). Such binding could improve the assembly of other OMPs indirectlyby eliminating misfolded polypeptides that could directly interferewith the assembly of normal polypeptide chains. Also, misfoldedpolypeptides could potentially sequester folding factors andthus indirectly influence the assembly of normal proteins, sotheir removal by DegP_S210A could prevent such a negative effecton overall assembly in the periplasm.

It can be argued that the overexpression of DegP_S210A failedto improve OmpC_2Cys assembly because the covalent disulfidebond formation would intrinsically disrupt normal assembly.Indeed, the absence of these bonds in a dsbA background allowsnormal OmpC_2Cys assembly (11, 30). However, the assembly ofOmpF mutants, in which the defect originates from noncovalentdisruptions, was also not improved by DegP_S210A overexpression(14). It thus remains to be seen whether DegP_S210A assists OMPassembly by acting as a general chaperone. Elegant genetic studiescarried out in Silhavy's lab showed that the synthetically lethalphenotype of degP::Tn10 surA::Km^r could be reversed by a plasmidexpressing protease-deficient DegP_S210A at a growth temperatureof $<=$ 37°C (19). Whether the observed reversibility by DegP_S210Ais through actually promoting assembly or sequestering misfoldedpolypeptides, as we have shown here, remained to be determined.

The recently solved crystal structure of protease-deficientDegP provides some exciting insights into the functioning ofthis protein (8). It suggests that partially folded polypeptidesmay be fed into DegP's central cavity which, in an open conformation,is accessible through the lateral pores. The presence of hydrophobicphenylalanine residues in the central cavity is likely to playan important role in substrate interaction. It is proposed thatat lower temperatures, when the protease activity is dormant,unfolded substrates are released from DegP for refolding. Inthe case of substrates such as OmpC_2Cys, where correct foldingmay be unattainable due to the covalent disulfide bond formation,the protein appears to be trapped eternally in this binding-releasecycle.

ACKNOWLEDGMENTS

We are grateful to Michael Ehrmann for providing plasmids expressingDegP_S210A and its His tag derivative. We thank Leanne Misrafor critically reading the manuscript.

This work was supported by a grant from the National Institutesof General Medical Sciences (GM48167). M.C. received funds fromthe Minority Graduate Education program.

FOOTNOTES

* Corresponding author. Mailing address: Molecular and Cellular Biology Program, Arizona State University, Tempe, AZ 85287-2701. Phone: (480) 965-3320. Fax: (480) 965-0098. E-mail: rajeev.misra{at}asu.edu.

REFERENCES

Top