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

Abstract

The expression of assembly-defective outer membrane proteins can 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 to the presence of two non-native cysteine residues, is lethal in cells lacking the major periplasmic protease, DegP. This lethality is not observed in dsbA strains that have diminished ability to form periplasmic disulfide bonds. Our data show that this OmpC_2Cys-mediated lethality in a degP::Km^r dsbA⁺ background can be reversed by a DegP variant, DegP_S210A, that is devoid of its proteolytic activity but retains its reported chaperone activity. However, DegP_S210A does not reverse the lethal effect of OmpC_2Cys by correcting its assembly but rather by capturing misfolded mutant OmpC polypeptides and thus removing them from the assembly pathway. Displacement of OmpC_2Cys by DegP_S210A also alleviates the negative effect that the mutant OmpC protein has on wild-type OmpF.

OmpC and OmpF represent a major class of Escherichia coli outer membrane proteins (OMPs). These proteins form water-filled channels that permit the diffusion of small solutes into the cell. Because of this property, OmpC and OmpF are called porins or sometimes “classical” porins, which refer to their stable β-barrel structure and nonspecific solute diffusion properties (17). Each porin monomer consists of a 16-stranded antiparallel β-barrel (22). These porins exist as homotrimers, in which three barrels assemble to produce three separate channels (22).

Besides their atomic structures, the assembly and targeting of the outer membrane β-barrel proteins have been an intense topic of study (for a recent review, please see reference 6). Based on studies conducted in several laboratories, it has been shown that the assembly of porin proteins begins with the proteolytic cleavage of an amino-terminal signal sequence that yields soluble nascent monomers. Rapid folding of these monomers is followed by their oligomerization into protease- and heat-sensitive trimers called metastable trimers, which slowly mature into protease- and heat-resistant trimers, the final product of assembly.

Although porins in vitro can be fully folded into their native trimeric structures from denatured monomers without the need of an accessory protein (7, 27), assembly in vivo is highly dependent on several cellular folding factors. Among them are specific periplasmic foldases such as SurA and FkpA, which catalyze cis-trans peptidyl-prolyl isomerization (16, 20), although it has never been demonstrated that this enzymatic activity is actually involved in OMP assembly. Consistent with this notion, Behrens et al. (3) have recently shown that SurA assists in OMP assembly independent of its isomerase activity. Another periplasmic foldase, DsbA, is involved in promoting correct disulfide bond formation of certain OMPs (2). The wild-type sequence of the classical porins, however, lacks cysteine residues and thus they assemble independently of DsbA's activity. In contrast to wild-type OmpC, the assembly of a mutant OmpC protein, OmpC_2Cys, containing two non-native cysteine residues (C74 and C154) is drastically influenced by DsbA (11). Since OmpC_2Cys is the subject of the present study, it is further discussed below.

Another periplasmic protein, Skp, is thought to assist in OMP assembly by acting as a general chaperone (5, 21). However, the skp-null mutant phenotype showing a dramatic reduction in OMP levels could not be repeated in other laboratories since it was first reported. Recent genetic studies have shown that the presence of skp- and surA-null alleles in a strain confers a synthetic lethal phenotype (19). Based on these studies, it has been proposed that Skp and SurA are components of separate folding pathways providing chaperone activities (19). Nonproteinaceous components, including phospholipids and lipopolysaccharide (which is 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 important when the machinery that ensures correct OMP assembly malfunctions or the primary sequence of an OMP endures an unacceptable alteration. One periplasmic protease, DegP, has received the most attention in connection with OMP assembly. degP-null mutants do not grow at temperatures above 39°C (10, 25, 26), presumably because of a greater need for the removal of misfolded proteins at elevated temperatures. However, expression of certain misfolded OMPs renders the DegP's presence essential even at lower growth temperatures (14). Elegant work carried out by Spiess et al. (25) showed that DegP, in addition to proteolysis, assists in the correct folding of a periplasmic protein. A recent genetic study also argued for DegP's involvement in providing periplasmic chaperone activities (19); however, this contention was not backed by direct biochemical evidence.

We have previously described the isolation of a novel class of OmpC mutants that affected either the channel or phage receptor property of the protein (13, 28). One such OmpC mutant carrying a R74C substitution functionally enlarged the channel to permit the entry of large sugars such as maltodextrins, which are normally excluded from the OmpC channel (13). This isolate was subsequently used for the isolation of phage-resistant mutants under conditions that demanded the retention of a functional OmpC channel but no phage receptor activity. A group of OmpC mutants from this selection contained a G154C substitution while retaining the original R74C alteration (11). This mutant OmpC protein, OmpC_2Cys, which now had two non-native cysteine residues in its mature sequence, conferred phage resistance due to a biogenesis defect (11).

The OmpC_2Cys biogenesis defect was shown to stem from the protein's ability to form disulfide bonds in DsbA⁺ cells (30); OmpC_2Cys assembles normally in a genetic background lacking DsbA. OmpC_2Cys produces a novel transdominant effect on OmpF (11). Since the effect on OmpF is specific, this suggests that either the inhibition was due to the mixing of mutant OmpC assembly intermediates with that of OmpF or that the two proteins share a common assembly factor.

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 with a diminished disulfide forming ability in the periplasm, the absence of DegP produced no lethal effects at growth temperatures of ≤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 that DegP_S210A alleviates OmpC_2Cys's lethal effects not by improving its folding or assembly but rather by sequestering misfolded mutant OmpC polypeptides and thus removing them from the normal assembly pathway.

MATERIALS AND METHODS

Media, reagents, and bacterial strains.

Luria broth medium, Luria agar (1.5% [wt/vol]) medium, and M63 salt-based minimal medium were prepared according to the method of Silhavy et al. (23). When cultures were grown on minimal medium, 0.4% glycerol and 0.1 mM amino acid mixture lacking methionine were added. When required, ampicillin (50 μg/ml), kanamycin (25 μg/ml), chloramphenicol (25 μg/ml), tetracycline (25 μg/ml), and IPTG (isopropyl-β-d-thiogalactopyranoside; 0.4 mM) were added to the growth medium. Heat-killed Staphylococcus aureus (Pansorbin) was purchased from Calbiochem. [³⁵S]methionine Expre protein labeling mixture was purchased from Du Pont-New England Nuclear. All other chemicals were of analytical grade. The bacterial strains used in the present study are listed in Table 1.

TABLE 1.

Strains used in this study

Strain	Relevant characteristicsa	Source or reference
MC4100	F−araD139 Δ(argF-lac)U139 rpsL150 flbB5301 ptsF25 deoC1 thi-1 rbsR relA	4
PLB3261	MC4100 ΔlamB106 φompC′::lacZ+	S. Benson
RAM412	PLB3261 ompC234 (OmpC1Cys) zei::Tn10 (50% linkage to ompC by P1 transduction)	12
RAM415	PLB3261 ompC402 (OmpC2Cys) zei::Tn10	12
RAM1015	MC4100 lamB677 (LamB amber)	Lab strain
RAM1025	RAM1015 degP::Kmr	Lab strain
RAM1120	RAM1025 pCS10 (DegPS210A)	This study
RAM1121	RAM1025 pACYC184	This study
RAM1122	RAM1015 pCS10	This study
RAM1123	RAM1015 pACYC184	This study
RAM1124	PLB3261 degP::Kmr pCS10	This study
RAM1125	RAM412 degP::Kmr pCS10	This study
RAM1126	RAM415 degP::Kmr pCS10	This study
RAM1127	RAM415 recA::Kmr	This study
RAM1128	RAM415 recA::Kmr pCS21 (DegPS210A-6His)	This study

^aKm^r, kanamycin resistance.

Pulse-chase labeling.

Pulse-chase labeling was performed essentially as described previously (14). Cells were grown overnight at 37°C in glycerol minimal medium supplemented with 0.1 mM amino acid mixture. After a 1-in-30 dilution of overnight culture into fresh medium supplemented with 0.4 mM IPTG, cells were grown to an optical density 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 of ice-cold 15% trichloroacetic acid. Cells were centrifuged at 12,000 × g for 5 min at 4°C. Pellets were washed once with 0.5 ml of 5% trichloroacetic acid and once with 95% ethanol and then dried under a vacuum. Dried pellets were resuspended in 200 μl of sodium dodecyl sulfate (SDS) buffer (20 mM Tris-HCl, pH 7.5; 10 mM EDTA; 1% SDS) and boiled for 5 min. Cell lysates (100 μl) were transferred into tubes containing 1 ml of the immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5; 20 mM NaCl; 1% Triton X-100) and MalE and LamB antibodies. Tubes were incubated overnight at 4°C on a rocker. To bring down immunocomplexes, heat-killed S. aureus cells (50 μl) were added, and tubes were incubated at 4°C on a rocker for at least 4 h. Immunocomplexes were recovered by centrifugation and washed twice in immunoprecipitation buffer, twice in Tris buffer (10 mM Tris-HCl; pH 7.5), and once in water. Washed pellets were resuspended in sample buffer, boiled, and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Gels were fluorographed, dried, and exposed to X-ray films at −85°C. Radioactive protein bands were quantified by using a Bio-Rad gel analyzer.

Membrane analysis.

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

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

Interaction of OmpC_2Cys with DegP_S210A.

In vivo interactions between OmpC_2Cys and DegP_S210A were examined by purifying the soluble His tag variant of DegP_S210A (DegP_S210A-6His) through Ni²⁺ affinity chromatography. Subsequently, fractions containing DegP_S210A-6His were examined for the presence of OmpC_2Cys by Western blot analysis. Briefly, 5 ml of cells containing pCS21 (in which the expression of DegP_S210A-6His is inducible by IPTG) were grown to an optical density at 600 nm of 0.2 in the presence of 0.4 mM IPTG. Cells were pelleted and resuspended in 2 ml of 10 mM imidazole containing 20 μl of freshly prepared lysozyme (100 mg/ml). Cells were lysed by a small French pressure cell and, after the removal of unlysed cells by low-speed centrifugation, lysates were centrifuged at 105,000 × g in a 50 Ti rotor for 1 h at 4°C to pellet the membranes. DegP_S210A-6His was purified from the supernatant by affinity chromatography in a Ni²⁺-nitrilotriacetic acid column according to the Pharmacia Biotech HisTrap purification protocol, except that the elution of DegP_S210A-6His was carried out in several steps involving a gradual increase in the imidazole concentration from 20 to 200 mM. The DegP_S210A-6His protein eluted with 200 mM imidazole.

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

Protein density levels from Coomassie blue-stained gels and Western blots were determined by using Scion Image Analysis software.

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-defective OmpF mutants is lethal in a DegP⁻ background (14). This is presumably because DegP degrades misfolded proteins, thus eliminating their toxic effects. As found for the assembly-defective OmpF mutants, the expression of OmpC_2Cys also demanded the presence of DegP for cell viability. Initially, this conclusion was reached due to our inability to introduce, through P1 transduction, the ompC402 (OmpC_2Cys) allele into a degP::Km^r strain. Subsequently, direct evidence for this synthetic lethality was obtained by demonstrating that the ompC402 allele could be transduced into a degP::Km^r background only when the expression of a protease-deficient DegP_S210A protein was simultaneously induced from a plasmid replicon (Fig. 1). In contrast, the parental ompC234 (OmpC_1Cys) allele was transducible into a degP::Km^r background at the expected frequency independent of DegP_S210A's expression (Fig. 1B).

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), it is unlikely that the rescue from OmpC_2Cys-mediated lethality by DegP_S210A involved degradation of the mutant protein. To substantiate this, we first confirmed that the overexpression of DegP_S210A did not directly or indirectly induce proteolysis of mutant envelope proteins. For this we utilized a LamB amber fragment whose in vivo degradation is largely dependent on DegP (Fig. 2). Pulse-chase experiments showed that overexpression of DegP_S210A did not increase the rate of degradation of the LamB amber fragment (Fig. 2). Instead, overexpression of DegP_S210A in a DegP⁺ background increased the half-life of the LamB amber fragment (Fig. 2), thus suggesting a protective rather than a destructive role for the variant DegP_S210A protein. This protective role of DegP_S210A was most apparent in a background devoid of wild-type DegP. These results clearly demonstrated that the overexpression of DegP_S210A did not involve increased proteolytic activity in vivo.

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⁺ (□), DegP⁻ (▪), DegP⁺/DegP_S210A (○), 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 mutant OmpC and wild-type OmpF. As found for the LamB amber fragment, the level of OmpC_2Cys in the envelope increased when DegP_S210A was overexpressed (Fig. 3). This result confirmed that the rescue from lethality did not involve degradation of the mutant protein. As mentioned earlier, the expression of OmpC_2Cys imposes a negative effect on the assembly of the wild-type OmpF protein, presumably by interfering with the oligomerization of OmpF monomers or sequestering a common assembly factor (11). Under conditions in which DegP_S210A overexpression elevated OmpC_2Cys levels, it also elevated OmpF levels, thus abolishing the negative effect of OmpC_2Cys on OmpF (Fig. 3, compare lanes 2 and 4).

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 increase in wild-type OmpF levels suggested that overexpression of DegP_S210 improved the envelope's assembly or folding environment. Consistent with this notion, it was demonstrated by Spiess et al. (25) that the protease-deficient DegP_S210A protein promoted proper folding of a periplasmic protein MalS. Also, based on genetic data, it has been recently argued that DegP may be involved as a general chaperone in the folding and assembly of OMPs (19). We therefore tested the possibility that the elevated OmpC_2Cys levels caused by DegP_S210A overexpression may be due to its improved assembly. For this we examined the solubility of membrane-bound OmpC in the anionic detergent Sarkosyl, which is known to solubilize inner membrane proteins and proteins that are loosely bound to the outer membrane. Since improperly assembled OMPs may not be correctly inserted in the outer membrane, they could be solubilized by Sarkosyl.

In the absence of DegP_S210A overexpression, the parental and mutant OmpC proteins fractionated with the membrane pellet from untreated envelopes (Fig. 4, lanes 1 and 2). However, when DegP_S210A was overexpressed, a significant portion of OmpC_2Cys, and to some extent OmpC_1Cys, became soluble even without detergent treatment (Fig. 4, lanes 3 and 4). When membranes obtained from cells not overexpressing DegP_S210A were treated with Sarkosyl, OmpC_2Cys, like the parental OmpC_1Cys protein, was present primarily in the Sarkosyl-insoluble membrane pellet (Fig. 4, lanes 5 and 6 [top panel]), suggesting that at least as far as detergent solubility is concerned it was properly inserted in the outer membrane. Detergent treatment of membranes obtained from DegP_S210A-overexpressing strains dislodged a large quantity of both OmpC species from the envelope (Fig. 4, lanes 7 and 8 [bottom panel]). These results showed that the DegP_S210A-mediated increase in OmpC levels did not lead to the proper assembly or insertion of OmpC in the envelope. This finding was further supported by the observation that the increase in the OmpC_2Cys level did not make cells sensitive to OmpC-specific bacteriophages, reflecting the continued presence of a conformationally defective OmpC_2Cys. The observation that overexpression of DegP_S210A causes the parental OmpC_1Cys protein to become detergent soluble suggests that porin assembly intermediates in general can be targeted by DegP_S210A. Consistent with this notion we found that a fraction of the assembly-competent wild-type OmpC protein was also subjected to DegP_S210A-mediated detergent solubilization (data not shown). The lack of this effect on OmpA by DegP_S210A overexpression may reflect distinct folding properties of its assembly intermediates compared to that of the trimeric porin proteins.

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 overexpression did not lead to its proper assembly or insertion, we were curious as to its localization in the cell envelope. This was examined by fractionating whole-cell envelopes into inner and outer membranes by using sucrose density gradients (Fig. 5). OmpC and OmpA (as a control protein) levels were quantified from the inner (buoyant densities of 1.15 to 1.16) and outer membrane (buoyant densities of 1.21 to 1.22) fractions (Table 2).

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 (○) that primarily localizes with the intermediate-density material are indicated.

TABLE 2.

Distribution of OmpC and OmpA in outer and inner membrane fractions

Relevant protein composition	Distribution of OmpC (%)		Distribution of OmpA (%)
	Outer membrane	Inner membrane	Outer membrane	Inner membrane
OmpC1Cys DegP+	94	6	91	9
OmpC1Cys DegPS210A	82	18	94	6
OmpC2Cys DegP+	92	8	90	10
OmpC2Cys DegPS210A	69	31	92	8

Remarkably, overexpression of DegP_S210A caused the localization of some OmpC_2Cys molecules to the inner membrane (Fig. 5) such that approximately four times more OmpC_2Cys was present there compared to that in a DegP⁺ background (Table 2). This increase in the inner membrane was not due to an overall increase in mutant protein levels because, in contrast to the inner membrane, the level of OmpC_2Cys decreased in the outer membrane (Table 2). Interestingly, the parental OmpC_1Cys protein was also subjected to this effect of DegP_S210A overexpression (Fig. 5), although a greater influence was observed for OmpC_2Cys (Table 2). Unlike these variant OmpC proteins, overexpression of DegP_S210A had no effect on OmpA's localization (Fig. 5 and Table 2); thus, this DegP_S210A-mediated relocalization was specific to the mutant proteins.

Interestingly, fractionation studies revealed that a significant portion of DegP_S210A was also localized in the inner membrane (Fig. 5). The inner membrane localization of DegP_S210A is consistent with a detailed fractionation analysis carried out previously by Skorko-Glonek et al. (24). The colocalization of DegP_S210A and OmpC in the inner membrane raises the possibility of direct interactions between these proteins. It is important to mention here that our fractionation studies showed that significant amounts of both DegP_S210A and mutant OmpC proteins were also present in the soluble fraction; the data showing enhanced solubility of mutant OmpC due to increased DegP_S210A expression are presented in Fig. 4. Experiments to examine a possible direct interaction between DegP_S210A and OmpC_2Cys from soluble fractions are described below.

Inner membrane-localized OmpC molecules have characteristics of unassembled polypeptides.

Properly assembled porin molecules are resistant to heat denaturation and detergent solubility. Heat sensitivity assays (Fig. 6) showed that the inner membrane-localized OmpC_1Cys and OmpC_2Cys denatured into monomers even when analyzed at room temperature (25°C). On the other hand, outer membrane-localized OmpC proteins displayed heat resistance that is characteristic of properly folded stable trimers (Fig. 6). Additionally, the inner membrane-localized OmpC molecules, in contrast to those localized in the outer membrane, could be completely solubilized by Sarkosyl (data not shown). Together, these results indicated that the inner membrane-localized OmpC molecules most likely represent unassembled polypeptides.

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_2Cys and a fraction of DegP_S210A in both the inner membrane and soluble fractions raised the possibility that these two proteins engage in direct interactions. To examine this, a variant DegP_S210A containing a His₆ tag was utilized. The His₆-tagged DegP_S210A protein (DegP_S210A-6His) was expressed in a background also expressing OmpC_2cys, and the soluble tagged DegP protein was purified through affinity chromatography with Ni²⁺ columns. Affinity-bound DegP_S210A-6His was eluted with imidazole, and the eluted material was tested for the presence of mutant OmpC.

Four equal aliquots of the fraction containing eluted DegP_S210A-6His were analyzed in separate lanes on a polyacrylamide gel and, after electroblotting, the filter was cut into strips corresponding to individual lanes and then hybridized with antibodies raised against DegP, OmpC, DegP plus OmpC, or OmpC plus OmpA (Fig. 7). The results showed unequivocally that the mutant OmpC_2Cys polypeptides coeluted with the DegP_S210A-6His protein (Fig. 7, lane 2). The absence of OmpA from the eluted DegP_S210A signified the specificity of the DegP_S210A-OmpC interaction (Fig. 7, lane 4). These results are consistent with the fractionation analysis (Table 2), which also showed a specific effect of DegP_S210A on OmpC_2Cys but not on OmpA.

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 of DegP_S210A relieved the toxicity of the mutant OmpC protein not by correcting assembly but rather by sequestering it. Capturing of the mutant OmpC_2Cys protein by DegP_S210A also alleviated the transdominant effect of mutant OmpC on OmpF's assembly (Fig. 3).

DegP and OMP assembly.

Both genetic (14, 15, 19) and biochemical (25) studies support the notion that DegP plays an important role in the assembly of envelope proteins. Initially, this was thought to be solely due to the degradation of mutant envelope proteins. However, the finding of Spiess et al. (25) suggested that DegP is also involved 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's chaperone activity for its assembly; thus, the notion that DegP can act as a “general” chaperone still awaits further biochemical verification.

The data presented here and published earlier (14) showed that a protease-deficient DegP_S210A, which retains its chaperone activity (25), stabilizes assembly-defective OMPs without actually improving their assembly. As demonstrated for OmpC_2Cys in the present study, the stabilization of mutant proteins likely involves their sequestration by the polypeptide-binding cavity of DegP_S210A (8). Such binding could improve the assembly of other OMPs indirectly by eliminating misfolded polypeptides that could directly interfere with the assembly of normal polypeptide chains. Also, misfolded polypeptides could potentially sequester folding factors and thus indirectly influence the assembly of normal proteins, so their removal by DegP_S210A could prevent such a negative effect on overall assembly in the periplasm.

It can be argued that the overexpression of DegP_S210A failed to improve OmpC_2Cys assembly because the covalent disulfide bond formation would intrinsically disrupt normal assembly. Indeed, the absence of these bonds in a dsbA background allows normal OmpC_2Cys assembly (11, 30). However, the assembly of OmpF mutants, in which the defect originates from noncovalent disruptions, was also not improved by DegP_S210A overexpression (14). It thus remains to be seen whether DegP_S210A assists OMP assembly by acting as a general chaperone. Elegant genetic studies carried out in Silhavy's lab showed that the synthetically lethal phenotype of degP::Tn10 surA::Km^r could be reversed by a plasmid expressing protease-deficient DegP_S210A at a growth temperature of ≤37°C (19). Whether the observed reversibility by DegP_S210A is through actually promoting assembly or sequestering misfolded polypeptides, as we have shown here, remained to be determined.

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