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J. Biol. Chem., Vol. 278, Issue 24, 22161-22167, June 13, 2003
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-Lactamase Is Independent of the Signal Recognition Particle*
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From the
Institute for Biochemistry and Molecular Biology, Hermann-Herder-Strasse 7, 79104 Freiburg, Germany,
Faculty of Biology, and Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg, Germany
Received for publication, January 28, 2003
, and in revised form, April 7, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-lactamase, a hydrolytic enzyme responsible for cleavage of the -lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in -lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and -lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of -lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of -lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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For a subset of integral membrane proteins, i.e. membrane proteins with large periplasmic domains, SRP and SecA cooperate during the integration process (8–10). Targeting of these proteins to SecY is exclusively mediated by SRP and FtsY, leading to a stable binding of ribosome-associated nascent chains to the translocon. Translocation of the periplasmic moiety across the inner membrane, however, requires the activity of SecA.
There is conflicting evidence as to the involvement of SRP in the targeting of a subset of secretory proteins like -lactamase, alkaline phosphatase (PhoA), or ribose-binding protein (11). The depletion of either FtsY or Ffh concomitantly leads to decreased translocation of these proteins, suggesting that SRP and its receptor are involved in their export across the inner bacterial membrane (11–13). Because these proteins are considered to be SecB-independent, it had been proposed that SRP functions as an export-specific chaperone rather than a targeting factor for -lactamase, PhoA, and ribose-binding protein, replacing SecB during export (11). The interpretation of in vivo Ffh and FtsY depletion experiments is, however, complicated by the observation that targeting and integration of SecY is SRP-dependent. Decreasing the cellular concentrations of either SRP or FtsY simultaneously reduces the concentration of active translocons, making it difficult to differentiate between those export defects caused primarily by SRP/FtsY depletion and those originating from diminished concentrations of SecY (6, 14).
In E. coli, the ability of SRP to interact with its substrate is predominantly dependent on the length and the hydrophobicity of the signal sequence (15–17). Signal anchor sequences of integral membrane proteins like mannitol permease (MtlA) (18) or FtsQ (17) have been shown to cross-link efficiently with SRP, whereas such an interaction cannot be detected with the cleavable signal sequences of secretory proteins such as the outer membrane protein OmpA (18). However, signal sequence mutants of the SecB-independent secretory protein PhoA can be cross-linked to SRP provided that the hydrophobicity of the signal sequence is increased by inserting multiple leucine residues (7, 15, 17). The SecB-dependent outer membrane protein PhoE has also been shown to interact with SRP under conditions in which the hydrophobicity and the extension of the 
-helical signal sequence is increased by replacing a helix-breaking glycine residue with a helix-promoting leucine residue (19). This PhoE derivative, however, is still exported in an SRP-independent manner. SRP binding to cleavable signal sequences of secretory proteins is further influenced by trigger factor, a ribosome-associated chaperone (15, 17, 18). Binding of trigger factor to OmpA nascent chains subsequently prevents SRP binding to OmpA. In the absence of trigger factor, however, SRP can be cross-linked to the OmpA signal sequence (18).
Among the SecB-independent secretory proteins, -lactamase has always been considered to be a bona fide SRP substrate, in particular because its signal sequence is more hydrophobic than signal sequences of other secretory proteins (7, 15). However, so far no detailed in vitro analyses have been performed to really study the involvement of SRP in the targeting pathway of -lactamase. Our analyses using protease protection assays, flotation gradient experiments, and chemical cross-linking demonstrate that targeting and export of -lactamase proceed independently of the SRP pathway.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Reagents—Growth media components and chemicals were obtained from Roth (Karlsruhe, FR Germany), Sigma, and Pierce. Restriction enzymes and other reagents for cloning and DNA purification were purchased from New England Biolabs (Frankfurt, FR Germany), Qiagen (Hilden, FR Germany), and Novagen (Schwalbach, Germany).
Cell Growth and General Techniques—E. coli strains for plasmid isolation were cultured in Luria Broth. For the preparation of inner membrane vesicles, cells were cultured in phosphate-buffered medium as described by Müller and Blobel (20). Cell extracts for in vitro transcription and translation were prepared from cells grown either on S30 medium (20) or on S150 medium (21, 22). When required, antibiotics were added to the culture medium at the following concentrations: 50 µg of ampicillin/ml, 25 µg of kanamycin/ml, and 10 µg of tetracycline/ml.
Plasmid Construction—pJMSKK, a kanamycin-resistant pBlue-script derivative, was constructed as follows. The ampicillin resistance gene (bla) of pBluescript SK+ was excised with BspHI and the vector blunt-ended with the Klenow fragment of DNA polymerase I. The bla gene was replaced with the kanamycin cassette from pFDX2322, cut with NaeI and SpeI, and also blunt-ended with the Klenow fragment of DNA polymerase I. pJMSKK was used to construct a vector carrying the bla gene under T7 promoter control. For this, the bla gene of pBluescript SK+ was excised with BspHI, blunt-ended with the Klenow fragment of DNA polymerase I, and ligated into the EcoRV site of pJMSKK. The constructed plasmid was termed pJMSKK Bla7. For the construction of the secA36 allele under T7-RNA polymerase control, genomic DNA of the SecA36 mutant GN42 was amplified using the primer SecA1 (5'-GAGATTTTCATATGCTAATCAAATTGTTAAC-3') and SecA2 (5'-CGCAGAATCCTCGAGCTTTTACTTCAACAG-3') introducing an NdeI and an XhoI cleavage site. After restriction digest the PCR product was cloned into the NdeI and XhoI sites of pET19b under the control of the T7 promoter. The resulting plasmid was termed pET19b-SecA36 and coded for a SecA36 derivative carrying an N-terminal His10 tag to expedite purification.
Purification of SecA, SecB, F1-ATPase, Ffh, FtsY, and SecA36 —The purification of SecA (23), SecB (22), F1-ATPase (24), Ffh (6), and FtsY (6) followed protocols reported previously. For the purification of His10-SecA36, pET19b-SecA36 was transformed into the E. coli strain TUNER(DE3) pLysS. Cells were grown at 37 °C overnight on Luria Broth supplemented with D-glucose (1%), washed, and subcultured on the same medium lacking glucose. At an A600 of 0.5, cells were induced with 1 mM isopropyl--D-thiogalactoside, incubated further until at an A600 of 1.5–1.8, and harvested. After resuspension in 50 mM triethanolamine acetate (pH 7.5), 50 mM KCH3COO (KOAc), 5 mM Mg(CH3COO)2, cells were disrupted using a French press at 8000 pounds/square inch in the presence of Complete protease inhibitor mixture (Roche Applied Science). After centrifugation at 30,000 x g, the supernatant was subjected to high speed centrifugation (150,000 x g), and SecA36 overproduction was confirmed by SDS-PAGE of the supernatant and subsequent Coomassie staining. For further purification the supernatant was buffer-exchanged in 2.5-ml steps on PD-10 columns (Amersham Biosciences), to adapt the salt conditions to 50 mM potassium phosphate (pH 7.5), and 300 mM KOAc, suitable for further purification using the TALON® IMAC System (Clontech Laboratories, Inc., Palo Alto, CA). The buffer exchanged supernatant was supplemented with Complete/EDTA protease inhibitor mixture and phenylmethylsulfonyl fluoride (0.5 mM), and 3.5 ml of this material was incubated overnight at 4 °C with 2 ml of the Talon resin. Washing was performed according to the manufacturer's manual with 50 mM potassium phosphate (pH 7.5), and 300 mM KOAc. After washing, the resin with the bound protein was filled into 2-ml gravity-flow columns (Qiagen, Hilden, Germany), and proteins were eluted stepwise with increasing imidazole concentrations (0–150 mM). SecA36 fractions were analyzed on a Coomassie-stained SDS gel, and pure SecA36 containing fractions were pooled. Protein concentrations were determined using the Pierce BCA protein assay kit.
In Vitro Reactions—The composition of the transcription/translation system of E. coli and the purification of its components, the preparation of INV, the flotation gradient analysis, and proteinase K protection assay employed in this study have been described previously (1, 6, 8). Synthesis of nascent chains was achieved as described in Beck et al. (18), in the presence of RNase H (1 unit/25 µl), 10 µg/ml 10Sa RNA antisense oligonucleotide (25), and 0.5 µg/µl oligonucleotide Bla-175 (5'-GCAGGCATCGTGGTGTCACG-3'). Chemical cross-linking using DSS (Pierce) was performed as described previously (18). The in vitro reactions for subsequent cross-linking experiments were performed in the presence of HEPES-NaOH instead of triethanolamine acetate. Immunoprecipitation was performed in 4–6-fold scaled up reactions using polyclonal rabbit antibodies against trigger factor and Ffh, covalently linked to protein A-Sepharose matrix (26).
Sample Analysis and Quantification—All samples were analyzed on SDS-polyacrylamide gels (13, 15, or 7–17%). Radiolabeled proteins were visualized by PhosphorImaging using a Amersham Biosciences PhosphorImager and quantified using ImageQuant software from Amersham Biosciences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-Lactamase—For analyzing its targeting to and its translocation across the E. coli membrane, the -lactamase gene of pBluescript II was cloned under a T7-dependent promoter in a kanamycin-resistant pBluescript derivative. In vitro synthesis was performed using a highly purified E. coli cell extract. Transport of -lactamase was analyzed using inside-out inner membrane vesicles (INV) and was compared with the transport of the SRP-dependent inner membrane protein mannitol permease (MtlA) and the SRP-independent outer membrane protein OmpA. As shown in Fig. 1, when synthesized in the absence of INV all three substrates were sensitive toward proteinase K digestion, but became protease-resistant in the presence of INV. Protease protection of both OmpA and -lactamase in the presence of INV was accompanied by signal sequence cleavage as indicated by the presence of a protease-resistant band of lower molecular mass. The lower molecular mass of the membrane-protected fragment of MtlA (MtlA-MPF) is due to the cleavage of the large C-terminally located cytoplasmic domain. The transport efficiency into INV of -lactamase is lower than the transport efficiency of OmpA, a phenomenon which has also been observed in vivo.
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A SecY Mutation Interfering with Efficient SecA Binding to the Translocon Blocks -Lactamase Transport—Although both the SRP-dependent and the SecA-dependent protein targeting pathways converge at the SecYEG complex in the inner bacterial membrane, we have shown recently (27) by using translocon mutants that different domains of SecY and different components of the translocon are engaged by both processes. These translocon mutants were assayed for their ability to transport -lactamase. As shown in Fig. 2 (top panel), the translocation of -lactamase is completely blocked in INV prepared from the secY mutants secY39 and secY205. A severe translocation defect is furthermore observed in INV from a secG deletion strain and in SecE-depleted INV. In these translocation assays using mutant INV, the effects of transport on -lactamase are indistinguishable from those of transport on the SRP-independent secretory protein OmpA (Fig. 2, middle panel) but clearly different from those on the integration of the SRP-dependent substrate MtlA (Fig. 2, bottom panel). The integration of MtlA is not significantly influenced by the secY205 mutation and the secG deletion. The biochemical characterization of the secY205 mutant had revealed that the translocation defect originates primarily from reduced SecA binding to the mutant SecY (28), and consequently, the transport of SecA-independent proteins like MtlA is not impaired by this mutation. The function of SecG is also specifically associated with the SecA function (29) and does not seem to be required for SecA-independent protein transport (27). The secY39 mutation affects both the SecA-dependent as well as the SRP-dependent protein transport. However, the effects on -lactamase and OmpA translocation are more severe than on MtlA integration. A complete block of MtlA integration can only be observed in SecE-depleted INV (Fig. 2, middle panel). SecE depletion concomitantly leads also to reduced SecY concentrations, because in the absence of SecE, SecY is rapidly degraded by the membrane-bound protease FtsH (30), resulting in a transport defect for both SecA- and SRP-dependent proteins. In summary, these data indicate that -lactamase translocation depends on the same domains of SecY and the same components of the translocon as the translocation of the SRP-independent substrate OmpA which argues for a SecA-dependent translocation of -lactamase.
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The SecA dependence was directly tested by utilizing a new assay system for SecA-dependent proteins, based on the biochemical complementation of the secY205 mutant INV with a mutant SecA (28). A systematic search for secA mutations suppressing the secY205 phenotype led to the isolation of allele-specific suppressors, one of which is the secA36 mutant, carrying a single amino acid substitution within the high affinity ATP-binding site of SecA (28). By using PCR we amplified the secA36 allele from the mutant strain GN42 and cloned it into a T7-dependent expression vector for overexpression and purification. The purified SecA36 was then tested for its ability to suppress in vitro the translocation defect of the secY205 INV. As shown in Fig. 3 (top panel), in the absence of SecA36, no -lactamase translocation into secY205 INV was observed; the presence of SecA36, however, enhanced -lactamase translocation significantly (Fig. 3, top panel, compare lanes 6 and 12). Purified wild type SecA, on the other hand, was unable to overcome the SecY205 defect (data not shown). Enhanced translocation of -lactamase in the presence of SecA36 was not only observed for secY205 INV but also for wild type INV, which is in agreement with the enhanced ATPase activity of SecA36. As a control, we also tested the effect of SecA36 on the translocation of the SecA-dependent OmpA (Fig. 3, lower panel). Like for -lactamase, the addition of SecA36 greatly enhanced the translocation of OmpA into secY205 INV and also into wild type INV. These data demonstrate that -lactamase, despite being a SecB-independent protein, requires SecA for translocation in the same way as the SecB-dependent protein OmpA.
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Ribosome-associated Nascent Chains of -Lactamase Are Not Cotranslationally Targeted to the SecY Translocon—In principle, the observation that export of -lactamase is catalyzed by SecA does not exclude an involvement of SRP during the targeting of -lactamase to the SecYEG complex. SRP-dependent targeting and SecA-dependent translocation as individual steps have been identified in the export of bacterial membrane proteins with large periplasmic domains (8, 10). One of the salient features of both the eukaryotic and prokaryotic SRP is that it interacts with its substrates cotranslationally and targets the ribosome-associated nascent chains (RNCs) via the SRP receptor to the translocon. SRP-dependent cotranslational targeting can be analyzed using flotation gradient centrifugation. Stable binding of RNCs to the translocon allows their selective recovery from the membrane fractions of such a flotation gradient. We have demonstrated recently (8) that this assay provides a reliable method to differentiate between SRP-dependent and SRP-independent substrates. Fig. 4A shows the results of flotation gradient centrifugation for ribosome-associated nascent chains of the SRP-independent substrate OmpA and the SRP-dependent membrane proteins MtlA and Momp2. Although the vast majority of OmpA RNCs was recovered from the bottom fractions 4 and pellet (P) of such a gradient, independent of whether INV were present or not, in the presence of INV MtlA and Momp2 nascent chains were mainly recovered from the membrane fractions 2 and 3 of the gradient, indicating cotranslational targeting. In flotation gradient centrifugation -lactamase RNCs behave clearly different from the MtlA and Momp2 RNCs in that even in the presence of INV 80% of the RNCs was recovered from the bottom fractions of the flotation gradient (Fig. 4B). We have shown recently that SRP is able to bind to the cleavable signal sequence of OmpA in the absence of the ribosome-associated chaperone trigger factor. We therefore analyzed targeting of -lactamase RNCs also in the absence of trigger factor. However, as shown in Fig. 4B, even in the absence of trigger factor no significant cotranslational binding of -lactamase RNCs could be observed, arguing against an SRP-mediated targeting of -lactamase RNCs.
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Chemical Cross-linking Reveals a Strong Interaction between -Lactamase RNCs and Trigger Factor but Not with SRP— Chemical and site-specific cross-linking has been used extensively to monitor the interaction between SRP and its substrates. In particular, by employing the membrane-permeable chemical cross-linker DSS, the individual steps of SRP binding to RNCs and their subsequent targeting to the translocon have been analyzed. This approach was utilized to determine directly whether SRP binds to -lactamase RNCs as they emerge from the ribosomal exit tunnel. -Lactamase RNCs were synthesized in a cell extract prepared from a wild type strain and a trigger factor deletion strain (
tig), both containing the endogenous Ffh concentrations. Samples were subsequently treated with DSS, and the identity of cross-linked bands was verified by immunoprecipitation with antibodies raised against Ffh and trigger factor. Under wild type conditions, a strong cross-link between -lactamase and trigger factor was observed (Fig. 5, lane 4), whereas no significant interaction with Ffh could be detected (Fig. 5, lane 3). We next considered the possibility that SRP might interact with -lactamase RNCs in the absence of trigger factor, a phenomenon that has been observed for OmpA RNCs (18). Synthesizing -lactamase RNCs in the tig cell extract and subsequent DSS cross-linking revealed only a very weak trigger factor interaction in comparison to the wild type conditions (Fig. 5, compare lanes 4 and 8), presumably resulting from trigger factor contaminations in our T7-RNA polymerase preparations, but more importantly, even in the absence of a strong trigger factor interaction, no enhanced Ffh binding to -lactamase was observed (Fig. 5, compare lanes 3 and 7). This was not due to limited amounts of Ffh present in the cell extract, because supplementation of the tig cell extract with purified Ffh during synthesis did not increase Ffh binding (Fig. 5, lane 11). In summary, chemical cross-linking revealed that SRP has only a weak affinity for -lactamase RNCs, which is most likely not sufficient to support SRP-dependent targeting.
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FtsY-depleted INV Support -Lactamase Translocation— The proposal that -lactamase is targeted to the membrane via the SRP pathway is mainly based on in vivo depletion experiments in which Ffh or 4.5 S RNA, the components of the bacterial SRP, or the SRP receptor FtsY had been depleted (11–13). In several studies (12) it had been shown that -lactamase export is sensitive toward reduced levels of SRP/FtsY suggesting a direct involvement of the SRP pathway in -lactamase transport. These studies, however, did not take into consideration that SecY, the central component of the bacterial translocon, is targeted to the membrane via the SRP pathway, and consequently any impairment of the SRP pathway will ultimately also affect the translocation of SRP-independent substrates. We therefore used an E. coli mutant strain in which the expression of FtsY is under the control of the araB promoter and monitored the cellular levels of both FtsY and SecY by Western blotting (data not shown). Cells were grown in the absence of arabinose and harvested immediately after the FtsY concentration declined but before a significant reduction of the SecY concentration was observed. These cells were then used for the preparation of INV.
We first tested the FtsY-depleted vesicles for their ability to support the integration of the SRP-dependent substrate MtlA. As shown in Fig. 6, MtlA integration was significantly impaired, demonstrating that the FtsY concentration in these vesicles is below the concentration required for efficient targeting of SRP-dependent substrates. Integration could, however, be restored by adding purified FtsY to these INV, indicating that the integration defect did not result from a reduced level of SecY translocons. This is further corroborated by the observation that the translocation of the SRP-independent substrate OmpA proceeds basically at wild type levels in the FtsY-depleted vesicles. The FtsY-depleted vesicles do not express any defect in the translocation of -lactamase, demonstrating that -lactamase transport, like the transport of OmpA, does not depend on FtsY. We also tried a similar approach to obtain INV from the conditional Ffh mutant strain WAM113 (data not shown). However, we were unable to reduce the cellular levels of Ffh without simultaneously reducing the cellular concentration of SecY. Consequently, INV from WAM 113 grown in the absence of arabinose failed to support even the translocation of the SRP-independent substrate OmpA. This underlines the difficulty in interpreting in vivo depletion studies using conditional SRP mutant strains.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-lactamase has been mainly deduced from in vivo depletion experiments, in which the individual components of the SRP pathway have been depleted, resulting in the accumulation of preproteins in the cytoplasm (11–13). A further characterization of SRP-dependent substrates in E. coli has, however, identified SecY as an SRP substrate, raising the possibility that the observed translocation defects are primarily the result of a reduced number of SecY translocons (6, 14). Notably, in these depletion experiments also SecB-dependent proteins like OmpF, LamB, and maltose-binding protein were affected (11, 12), further pointing to secondary effects obscuring the true substrate specificity of the bacterial SRP. This has been confirmed by recent in vivo analyses using a temperature-sensitive ffh mutant (32). Although the insertion of the membrane proteins FtsQ and AcrB was significantly impaired in this mutant, there was no effect on signal sequence processing of secretory proteins like PhoA, maltose-binding protein, or LamB. The processing of -lactamase, however, was slightly delayed under non-permissive conditions (32). These data could indicate that among the secretory proteins, at least -lactamase might interact with SRP. This has been also deduced from the fact that the signal sequence of -lactamase is more hydrophobic than the signal sequences of other secretory proteins (Table II) (7).
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By using protease protection assays, flotation gradient centrifugation, and chemical cross-linking, we provide a detailed in vitro analysis of -lactamase export, demonstrating that in contrast to previous proposals the export of -lactamase does not depend on the SRP-targeting pathway. These results are important in view of the question of what are the determinants for substrate recognition by the bacterial SRP and, in particular, whether the bacterial SRP pathway is also involved in the export of secretory proteins. In several studies (15–17) it has been shown that the hydrophobicity of the targeting signal is probably the dominating feature for the selective recognition by the bacterial SRP. SRP is able to bind to mutated signal sequences of secretory proteins, characterized by increased hydrophobicity (7, 15, 17, 33, 34). Binding of SRP to this modified signal sequence, however, does not necessarily route the protein into the cotranslational targeting pathway. A PhoE mutant, carrying a glycine to leucine substitution within the signal sequence, is bound with high affinity by SRP as analyzed by chemical cross-linking but does not depend on the SRP pathway in vivo (19). Calculating the hydrophobicity of different signal sequences from secretory proteins and the first signal anchor sequences of membrane proteins using the MEMSAT algorithm (35) reveals that the hydrophobicity value of both -lactamase and the membrane protein leader peptidase are within the cut-off range of 2.8–3.2 for which the overlap between predicted membrane proteins and predicted soluble proteins is minimized (Table II). The hydrophobicity values of other experimentally identified SRP substrates are constantly above this cut-off value, and the value for secretory proteins is always below. This supports the argument that the hydrophobicity is the main determinant for SRP binding but further suggests that for proteins with intermediate hydrophobicity other factors might be important as well for selecting the targeting route.
We have shown previously (18) that the secretory protein OmpA is recognized by SRP in the absence of the ribosome-associated chaperone trigger factor, suggesting that secretory proteins, as they emerge from the ribosome, are preferentially recognized by trigger factor, preventing a stable interaction with SRP. This is different from what we observed for -lactamase nascent chains. Even in the absence of trigger factor, only a weak interaction with SRP can be detected. This is surprising in view of the significantly higher hydrophobicity of the -lactamase signal sequence in comparison to the OmpA signal sequence (Table II). Thus, neither the hydrophobicity of the -lactamase signal sequence nor binding of trigger factor to -lactamase provides a comprehensive explanation as to why SRP fails to interact with -lactamase RNCs.
It has been suggested recently that the conformation of the signal sequence is an important feature for the recognition by the bacterial SRP. Studies with PhoE signal sequence mutants indicate that the presence of a helix-breaker like proline prevents the recognition by SRP (19), suggesting that besides the hydrophobicity the length of the -helical structure of a signal sequence is an important feature for selecting the targeting route. A similar observation has also been made for the substrate recognition by the SRP of higher eukaryotes. Mutated signal sequences of invertase, carrying helix-breaking amino acids within the hydrophobic core, do not interact with the mammalian SRP (36). Although helix-destabilizing amino acids are frequently found toward the C terminus of the hydrophobic core of cleavable signal sequences (19), the hydrophobic core of the -lactamase signal sequence contains two proline residues, one of which is located within the center of the hydrophobic core, and this probably has an even more pronounced destabilizing effect on the -helical properties of the signal sequence, subsequently preventing a stable interaction with SRP.
Collectively, our results demonstrate that the SecB-independent protein -lactamase does not interact with the bacterial SRP and can be efficiently exported in the absence of a functional SRP pathway. It has been shown previously that -lactamase interacts with GroEL and GroES (37, 38) and that export of -lactamase is impaired in groEL and groES mutants (39). It therefore seems to be likely that GroEL and GroES substitute for SecB as transport-specific chaperones. Furthermore, the data presented here strongly support the view that the bacterial SRP is specific for membrane proteins and is not involved in the export of secretory proteins.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 49-761-2035250; Fax: 49-761-2035253; E-mail: Hans-Georg.Koch{at}biochemie.uni-freiburg.de.
1 The abbreviations used are: SRP, signal recognition particle; INV inner membrane vesicle; PhoA, alkaline phosphatase; Bla, -lactamase; MtlA-MPF, membrane-protected fragment of mannitol permease; RNCs, ribosome-associated nascent chains; DSS, disuccinimidyl suberate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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