From the Department of Molecular Microbiology, Vrije
Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
and the § Genetics and Biochemistry Branch, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892-1810
Received for publication, November 14, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Hemoglobin protease (Hbp) is a
hemoglobin-degrading protein that is secreted by a human pathogenic
Escherichia coli strain via the autotransporter mechanism.
Little is known about the earliest steps in autotransporter secretion,
i.e. the targeting to and translocation across the inner
membrane. Here, we present evidence that Hbp interacts with the signal
recognition particle (SRP) and the Sec-translocon early during
biogenesis. Furthermore, Hbp requires a functional SRP targeting
pathway and Sec-translocon for optimal translocation across the inner
membrane. SecB is not required for targeting of Hbp but can compensate
to some extent for the lack of SRP. Hbp is synthesized with an
unusually long signal peptide that is remarkably conserved among a
subset of autotransporters. We propose that these autotransporters
preferentially use the co-translational SRP/Sec route to avoid adverse
effects of the exposure of their mature domains in the cytoplasm.
Hemoglobin protease
(Hbp)1 is secreted by a human
pathogenic Escherichia coli strain (1) and contributes to
the pathogenic synergy between E. coli and Bacteroides
fragilis in intra-abdominal infections (2). It represents the
first described member of the serine protease autotransporters of
Enterobacteriaceae (SPATE) group of autotransporter proteins (3).
The key feature of an autotransporter is that it contains all the
information for secretion in the precursor of the secreted protein
itself (3). Autotransporters comprise three functional domains: 1) an
N-terminal targeting domain; 2) a C-terminal translocation domain; and,
in between these two, 3) the passenger domain that is the actual
secreted moiety. The C-terminal domain is supposed to form a
Compared with other signal peptides in E. coli, the putative
signal peptide of Hbp is unusually long (1) (Fig.
1). Analogously, several other
autotransporters are predicted to have long signal peptides (3, 5). All
these signal peptides display a conserved domain structure. The C
terminus resembles a normal signal peptide with a basic N-terminal
region, a hydrophobic core region, and a C-terminal consensus signal
peptidase cleavage site. The N terminus forms a conserved extension,
the function of which is not known (Fig. 1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel structure in the outer membrane that may form an oligomeric
channel around a cavity to allow the passage of the passenger
domain (4). The N-terminal domain is thought to function as a signal
peptide to mediate targeting to and translocation across the inner membrane.
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Fig. 1.
The signal peptide of Hbp has a conserved
N-terminal extension. A schematic representation of the 52 amino
acid-long signal peptide of Hbp is shown, indicating the probable
signal peptidase cleavage site (arrow). The basic N-terminal
(N), hydrophobic (H), and C-terminal
(C) domains characteristic of typical signal peptides are
indicated. M indicates the beginning of the mature region of
Hbp. A comparison of the N-terminal extensions of several
autotransporters possessing extended signal peptides is given together
with a consensus sequence of the conserved domain.
Most periplasmic and outer membrane proteins synthesized with a cleavable signal peptide are translocated through the Sec-translocon. The core translocase consists of the integral inner membrane proteins (IMPs) SecY, SecE, and SecG, which constitute an oligomeric complex homologous to the Sec61 channel complex in the endoplasmic reticulum (6). The peripheral membrane ATPase SecA is unique to bacteria and catalyzes the actual polypeptide transfer through the translocase. Targeting to the Sec-translocon may occur after translation and often requires the cytosolic chaperone SecB.
The Sec-translocon is also used for the membrane insertion of most IMPs that are synthesized with uncleaved, relatively hydrophobic signal peptides (7). Targeting of IMPs to the Sec-translocon is not mediated by SecB but by the signal recognition particle (SRP) and its receptor, FtsY, in a co-translational mechanism that resembles targeting to the Sec61 complex in the endoplasmic reticulum (8).
Here we provide evidence that the long signal peptide of Hbp mediates
targeting to the inner membrane via the SRP pathway. When the SRP
pathway is compromised, SecB can prevent, to a certain extent, the
mislocalization of pre-pro-Hbp. Subsequent translocation across the
inner membrane involves the Sec-translocon. This is the first
demonstration of the use of the co-translational SRP pathway for inner
membrane targeting of an extracellular protein.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains, Plasmids, and Media-- E. coli K12-strains and the plasmids used are listed in Table I. E. coli strains were routinely grown in Luria-Bertani (LB) medium (9). Strains MM152 and HDB52 were grown in M9 medium (9). If required, antibiotics were added to the culture medium.
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Reagents and Sera--
Restriction enzymes, the Expand long
template PCR system and the Lumi-light Western blotting substrate were
obtained from Roche Molecular Biochemicals. All other chemicals were
supplied by Sigma. Antiserum J40 raised against purified Hbp has been
described previously (2). Antisera against -lactamase, OmpA/OmpC,
trigger factor (TF)/SecA, and SufI were gifts from J.-M. van Dijl,
J.-W. de Gier, W. Wickner, and T. Palmer, respectively.
Plasmid Construction--
For cross-linking of nascent Hbp, we
constructed pC4MethssHbp, which encodes the 110 N-terminal amino acid
residues of Hbp fused to a C-terminal 4× methionine tag and a three
amino acid linker sequence to improve labeling efficiency. The
construct was obtained by PCR using pHE12.6 as a template and
the primers Hbp-EcoRI-for
(5'-GCCGGAATTCTAATATGAACAGAATTTATTCTCTTC-3' with the
EcoRI restriction site in boldface) and
Hbp-BamHI-rev (5'-GCGGGATCCACCGATTTCCGAATCCACA-3'
with the BamHI restriction site in boldface). The PCR
fragment was cloned EcoRI/ BamHI into pC4Meth
(10). To construct plasmid pHB6.4-Hbpss, the signal peptide coding
region of hbp was removed from plasmid pHB6.4-Hbp using the
Exsite PCR-based, site-directed mutagenesis kit (Stratagene). The
plasmid pHB6.4-Hbp is derived from pHE12.6 (1). The primers used were
Hbp-NheI-for
(5'-CTTGCTAGCGTCAATA-ATGAACTCGGGTATC-3' with the
NheI restriction site in boldface) and
Hbp-BglII-rev (5'-CTGATTTTATTTTTCTCAGGAGTAATTAAAAATGAAGAGATCTAAG-3'
with the BglII restriction site in boldface). Upon
ligation of the linear DNA, the last three bases from each end
constitute a HindIII restriction site. The resulting plasmid
encodes the Hbp protein without its signal peptide but with six extra
N-terminal amino acids (MKRSKL) and two altered amino acids (GT
AS)
at the start of the mature Hbp region.
In Vitro Transcription, Translation, Targeting, and Cross-linking-- Truncated mRNA was prepared as described previously (11) from HindIII linearized pC4MethssHbp. In vitro translation, targeting to inverted membrane vesicles (IMVs), cross-linking with DSS, and carbonate extraction of nascent Hbp were carried out as described (11). The samples were either analyzed directly by 15% SDS-PAGE or immunoprecipitated first using 4-fold the amount used for direct analysis.
Pulse-Chase Experiments--
For Ffh depletion studies, strains
HDB51 and HDB52 were grown overnight in M9 containing 0.1%
casaminoacids (Difco), 0.2% fructose, and 0.2%
L-arabinose, washed in the same medium lacking L-arabinose, and diluted to an OD660 of 0.004 in M9 containing fructose (0.2%) and either L-arabinose
(0.2%) or glucose (0.2%). Cells were grown to an OD660 of
~0.3 before labeling. Depletion of Ffh was verified by Western
blotting. The temperature-sensitive SecY mutant strains IQ85 and its
isogenic wild type strain IQ86 were grown overnight at 30 °C in LB
medium, diluted into fresh medium to an OD660 of 0.02, and
grown to an OD660 of 0.3. Growth was then continued at 30 or 42 °C for 3 h. For overexpression of a dominant lethal
ftsY allele, strain BL21(DE3) harboring pET9-FtsY-A449 was
grown overnight at 37 °C in M9 medium with 0.2% glycerol as a
carbon source and diluted into fresh medium to an OD660 of
0.03. When cells reached an OD660 of 0.2, 40 µM isopropyl-1-thio--D-galactopyranoside was added to induce FtsY-A449 expression, and growth was continued for
15 min. To inhibit SecA functioning in MC4100, 3 mM
NaN3 was added 3 min prior to labeling. In all experiments,
2.4 OD660 units of cells were washed and diluted into 3 ml
of M9 medium containing appropriate sugars and a mixture of 18 amino
acids except methionine and cysteine. After recovery for 15-45 min at
30, 37, or 42 °C as indicated, cells were pulse labeled for 1 min by
the addition of 10 µCi/ml [35S]methionine and chased
for various times by adding cold methionine (2 mM). To stop
the chase, cells were rapidly cooled in ice water and centrifuged at
4 °C for 2 min at 8,000 × g. Supernatants were precipitated with trichloroacetic acid and subjected directly to
8-15% SDS-PAGE. Cell pellets were first lysed and subjected to
immunoprecipitation using anti-Hbp and anti-OmpA serum essentially as
described (12).
Western Blotting-- For analysis of steady-state levels of Hbp, E. coli strains harboring an hbp expression plasmid were grown to an OD660 of 0.5. Aliquots were removed from the cultures and centrifuged (1 min at 16,000 × g). The culture supernatants were trichloroacetic acid precipitated. Equivalent amounts of cells and supernatant were analyzed by Western blotting. Blots were developed by enhanced chemiluminescence using Lumi-light Western blotting substrate.
Sample Analysis--
Radiolabeled proteins were visualized by
phosphorimaging using a Amersham Biosciences PhosphorImager 473 and quantified using the ImageQuant quantification software from
Amersham Biosciences. Chemiluminescent Western blots were analyzed
using the Fluor-S MultiImager and Multianalyst software (Bio-Rad).
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RESULTS |
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Hbp That Lacks Its Signal Peptide Is Not Secreted and Degraded in
the Cytosol--
Hbp is synthesized in a pre-pro form (148 kDa) (1).
The N-terminal signal peptide is cleaved during passage of the
pre-pro-Hbp through the inner membrane, leaving the pro-Hbp (142 kDa)
in the periplasm. The C-terminal -barrel domain is cleaved from the pro-Hbp at the outer membrane and mediates the transfer of the mature
Hbp (111 kDa) into the culture medium. To analyze the role of the Hbp
signal peptide, it was deleted from the pre-pro-Hbp, and the
consequences for maturation and secretion were analyzed by Western
blotting of cell samples and culture supernatants using Hbp-specific
antibodies (Fig. 2A). As
expected, deletion of the signal peptide (
ss) prevented secretion of
mature Hbp into the medium. Only a small amount of pro-Hbp
ss was
detected in the cells. In contrast, most wild-type pre-pro-Hbp was
processed to mature Hbp, which was either secreted or remained
cell-associated as observed previously (1). In addition, some pre-pro-
or pro-Hbp accumulated in the cells.
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We next investigated the reason for the low expression level of
pro-Hbpss. It has been observed previously that secreted proteins
and IMPs that fail to be translocated across or inserted into the inner
membrane are prone to proteolytic degradation (13). To investigate this
possibility, Hbp
ss was expressed in strain HDB107, which lacks the
major cytoplasmic proteases Lon and ClpYQ (13). Pulse-chase labeling
was employed to compare the stability of Hbp
ss species in strain
HDB107 and its isogenic parental strain HDB97 (Fig. 2B).
Hbp
ss remained almost completely stable for at least 10 min in
HDB107, whereas only limited amounts were detected in HDB97 after a
10-min chase. In fact, relatively little Hbp
ss was detected in HDB97
even when samples were analyzed directly after pulse labeling.
These data indicated that Hbp requires its signal peptide for targeting to the inner membrane and, consequently, for secretion of mature Hbp. Mislocalized pro-Hbp is rapidly degraded by the cytoplasmic proteases Lon and/or ClpYQ.
Nascent Hbp Interacts with SRP, Trigger Factor, SecA, and SecY in Vitro-- To investigate the molecular interactions of the atypical Hbp signal peptide in the cytoplasm and the membrane, we used an in vitro cross-linking assay. A radiolabeled Hbp translation intermediate of 117 amino acid residues was generated by in vitro translation of truncated mRNA in a homologous cell-free translation system developed previously in our laboratory (11). Because the truncated RNA does not contain a stop codon, the nascent chain remains associated with the ribosome, and the signal peptide is exposed outside the ribosome that covers ~35 C-terminal amino acids. Translation was carried out in the presence of purified IMVs to allow targeting and membrane insertion. Subsequently, interactions of the nascent Hbp were fixed by using the membrane-permeable, lysine-specific cross-linking reagent DSS. Finally, the samples were extracted with sodium carbonate to separate membrane integrated from the soluble and peripheral membrane proteins.
Approximately 30% of the synthesized nascent Hbp was detected in the
carbonate pellet (Fig. 3, quantification
data not shown). Given the relatively low intrinsic efficiency of the
E. coli in vitro translocation system,
this result indicated that nascent Hbp is properly targeted and
inserted into the membrane and remains anchored via its signal peptide
that is not cleaved at this nascent chain length. In the untargeted
(carbonate soluble) fraction, a cross-linking product of ~60 kDa
appeared that could be immunoprecipitated using serum directed against
Ffh, the protein component of the SRP (Fig. 3, lane 4). The
molecular mass of this product is consistent with the combined
molecular mass of Ffh (50 kDa) and the Hbp 117-mer (13 kDa). The
cross-linking to Ffh is remarkably strong considering the low abundance
of the SRP in the translation lysate, suggesting that it represents a
functional interaction. Weaker cross-linking products of slightly
higher molecular mass were immunoprecipitated using anti-TF serum (Fig.
3, lane 3). TF (54 kDa) cross-linked to nascent chains often
migrates at varying positions (10). The membrane-integrated nascent Hbp
was primarily cross-linked to SecA (102kDa) (Fig. 3, lane
7). In addition, a very faint cross-linking smear was observed at
~46 kDa that contained SecY, as evident from long exposures of
immunoprecipitated samples (not shown).
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Together, the cross-link patterns are reminiscent of those found with the nascent IMPs FtsQ and leader peptidase I (Lep) (10, 11, 14). Both FtsQ and leader peptidase I are targeted by the SRP to the Sec-translocon (15, 16). Apparently, nascent Hbp can be targeted to the inner membrane and inserts close to the Sec-translocon. The unprecedented strong cross-linking to Ffh, considering the extremely low abundance of Ffh in the translation lysate, suggests a high affinity of the Hbp signal peptide for the SRP and, consequently, a role for the SRP in the targeting of Hbp.
Hbp Requires the SRP for Optimal Processing and Secretion in Vivo-- We investigated whether the interaction of nascent Hbp with the SRP observed in vitro reflects a dependence on the SRP targeting pathway for processing and secretion of the full-length protein in vivo. Strains that are conditional for the expression of targeting factors were used in pulse-chase experiments to analyze the effects on the kinetics of processing as described above. Furthermore, spent medium of the pulse-chase samples was analyzed to monitor the secretion of mature Hbp in time.
Under normal conditions, N-terminal processing appeared to be very
fast, and pre-pro-Hbp could only be detected in the pulsed sample (Fig.
4A, lane 7) as has
been observed for many pre-secretory proteins such as OmpA (see also
Fig. 4C, lane 4). However, C-terminal processing
of Hbp is much slower. Under the expression conditions used, not all
pro-Hbp was converted into mature Hbp even after 1 h of chase
(Fig. 4A, lane 12). The actual release of mature Hbp into the culture medium is even slower, appearing prominently only
after 1 h of chase (Fig. 4B, lane 12).
Depletion of Ffh resulted in an accumulation of pre-pro-Hbp in the
cells (Fig. 4A, left panel). The
amount of secreted mature Hbp after 60 min of chase, but not the
kinetics of the secretion of mature Hbp, appeared affected upon
depletion of Ffh (Fig. 4B, left
panel). As a control, the processing of OmpA was hardly
influenced (Fig. 4C, left panel), which is in
agreement with its requirement for SecB rather than SRP for targeting
(17). In an alternative approach to analyzing the role of the
SRP-targeting pathway in Hbp secretion, the effect of overexpression of
FtsY-A449 was investigated. This mutant SRP receptor has a reduced
GTP-binding capacity as a result of an amino acid substitution in the
fourth GTP-binding consensus element (18). Moderate overexpression of
FtsY-A449 has been shown to compromise SRP-mediated protein
targeting (18). Hbp processing and, consequently, also secretion
appeared to be impaired upon moderate overexpression of FtsY-A449 as
opposed to the non-induced expression level (Fig. 4, D
and E). OmpA processing was not affected under these
conditions (Fig. 4F).
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To examine whether the other major targeting factor, SecB, is also involved in Hbp targeting, Hbp was expressed in strain MM152, which lacks SecB, and in its isogenic wild-type strain, MC4100. Pre-pro-Hbp did not accumulate in the SecB minus strain, nor was the secretion of mature Hbp affected (Fig. 4, G and H). As a control, pre-OmpA accumulated at early chase times in strain MM152. Apparently, SecB is not necessary for efficient targeting of Hbp per se.
We next considered the possibility that the residual processing and secretion of Hbp under Ffh-deficient conditions is due to alternative targeting via SecB. Consistent with this explanation, the expression of Hbp in a double mutant strain (SecB knockout, Ffh conditional) showed a much stronger secretion phenotype upon Ffh depletion than the single (Ffh conditional) mutant (Fig. 4, J and K, left panels). The secretion defect was greatly reduced in cells that express Ffh (Fig. 4, J and K, right panels), again suggesting that SecB is not required for the targeting of Hbp under normal conditions. The accumulation of pre-pro Hbp under these conditions at early chase times remains unexplained but may be related to adverse effects of the unnatural control of Ffh expression from the arabinose promoter. Interestingly, a similar but opposite additive effect is observed for OmpA. When the preferred targeting factor, SecB, is absent, processing is impaired (Fig. 4L, right panel). Additional depletion of Ffh completely blocks residual OmpA processing (Fig. 4L, left panel).
Together, these results suggested that Hbp requires the SRP pathway for optimal targeting to the inner membrane. Although SecB is not essential for targeting per se, it can apparently compensate to a certain extent for depletion of the SRP.
Hbp Requires SecA and SecY for Efficient Processing and Secretion in Vivo-- Two main types of translocons mediate the transfer of proteins across the inner membrane, namely the Sec-translocon (6) and the Tat-translocon (19). We investigated the role of these translocons in the secretion of Hbp in vivo using the pulse-chase approach described above.
A temperature-dependent conditional secY mutant
strain was used to deplete the cells for functional Sec-translocons. At
the non-permissive temperature, processing of the control Sec substrate OmpA was severely impaired in this strain as compared with its parental
wild-type strain (Fig. 5C).
Likewise, pre-pro-Hbp accumulated in the secY Ts cells (Fig.
5A, left panel), indicating that
translocation of Hbp across the inner membrane proceeds through the
Sec-translocon.
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To study the role of SecA, its ATPase activity was perturbed with azide. Under the conditions used, processing of OmpA was almost completely inhibited (Fig. 5F, left panel). Again, pre-pro-Hbp accumulated in the cells, indicating that translocation of Hbp is dependent on SecA (Fig. 5A, left panel). A similar dependence on functional SecA was observed when a temperature-dependent conditional secA mutant strain was used (data not shown).
The Tat-translocon is used by a subset of preproteins that are folded prior to translocation (19). Tat substrates carry an essential twin arginine motif in their signal peptide just upstream of the hydrophobic domain. Although the Hbp signal peptide does not fully comply with this motif, it does contain two consecutive arginine residues upstream of the hydrophobic core region. This feature prompted us to investigate a possible role of the Tat-translocon using a strain that lacks TatA and TatE, rendering it completely unable to translocate Tat substrates. This double mutant strain showed normal kinetics of processing and secretion of Hbp whereas processing of the known Tat substrate SufI was completely blocked, arguing that the Tat-translocon is not involved in secretion of Hbp (data not shown).
Together, the data suggest that Hbp uses the Sec-translocon for
transfer across the inner membrane consistent with the in vitro cross-link data (Fig. 3). SecA appears to be required to drive the translocation process.
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DISCUSSION |
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In the present work, we have addressed the question how the autotransporter Hbp is targeted to and translocated across the inner membrane. Both in vitro cross-linking and in vivo pulse-chase labeling experiments point to the use of a co-translational targeting and translocation mechanism involving the targeting factor SRP and the Sec translocation machinery. This is the first example of an extracellular protein that can be targeted by the SRP. Interestingly, in the absence of a functional SRP pathway, part of the mistargeted Hbp is rescued by SecB, underscoring the inherent flexibility of protein targeting in E. coli (20).
What are the features in the Hbp signal peptide that determine SRP binding? Previous work in our group has demonstrated that the SRP preferentially interacts with relatively hydrophobic signal peptides such as those that are present in IMPs (10). However, the hydrophobic core region at the C terminus of the Hbp signal peptide is not particularly hydrophobic. Interestingly, the Hbp signal peptide is relatively long (52 amino acids) and appears to contain an N-terminal extension that precedes a "classical" signal peptide (Fig. 1). It is attractive to speculate that the N-terminal extension plays a role in the recognition by the SRP either directly or indirectly by presenting the hydrophobic core in a favorable conformation or by recruiting other factors that increase the affinity of the Hbp signal peptide for the SRP. It is worth mentioning that the only other known example of a secreted protein that makes use of the SRP for targeting, SecM, is also synthesized with a long signal peptide that comprises an N-terminal extension and a moderately hydrophobic core region (21). SecM is a regulatory protein that functions in the secretion-responsive control of SecA expression. In wild-type cells, SecM is translocated to the periplasm where it is rapidly degraded (22).
Alternatively, the N-region (KCVHKSVRR) between the hydrophobic core and the N-terminal extension might be important for SRP recognition of the Hbp signal peptide. Compared with other signal peptides in Gram-negative bacteria, this region is more basic. Interestingly, the crystal structure of the SRP has revealed an unusual RNA-protein interface that is thought to constitute the signal peptide binding groove (23). It has been suggested that the protein moiety of the interface interacts with the hydrophobic core of the signal peptide, whereas the RNA is responsible for recognizing the basic N-domain. Following this reasoning, a more basic N-domain might compensate for a less hydrophobic core region in SRP binding. These possibilities are currently being investigated.
Translocation of autotransporters across the inner membrane has been proposed to involve the N-terminal signal peptide and occur via the Sec-pathway, which is also used by periplasmic and outer membrane proteins (3). Consistent with this proposal, our data suggest that the Sec-translocon receives and translocates the nascent Hbp. In this respect, Hbp resembles IMPs like leader peptidase I, FtsQ, and MtlA (7). An accessory translocon component, YidC, is specifically involved in membrane integration of these IMPs but not in the translocation of secretory proteins (11, 24).2 We have not observed any cross-linking of nascent Hbp with YidC. Moreover, depletion of YidC did not affect processing and secretion of Hbp (data not shown). Apparently, YidC is dispensable for the reception of Hbp at, as well as the translocation of Hbp across, the inner membrane-embedded Sec-translocon.
It is not unlikely that other members of the autotransporter family
follow the same pathway of targeting and translocation across the
bacterial inner membrane. Many autotransporters carry signal peptides
of similar length and domain structure (3). The N-terminal extension in
these signal peptides is remarkably conserved, as is the basic
character of the N-domain. In addition, substrates of an analogous
secretion system, the "two-partner secretion" (TPS) pathway in
which the -barrel domain is present in a separate protein, also
possess signal peptides that are conserved with members of the
classical autotransporter family (5). One of these substrates, the HMW1
adhesin from Haemophilus influenzae that carries a 68 amino
acid-long signal peptide was shown to require SecA and SecE for
maturation and secretion (25).
What would be the benefit of co-translational translocation for
autotransporters? For Hbp, it might prevent degradation or premature
folding of Hbp in the cytoplasm. Hbp that lacks its signal peptide
appeared to be vulnerable to degradation by cytoplasmic proteases. It
should be noted that both the autotransporter and two-partner secretion
families comprise many virulence factors such as hemagglutinins,
hemolysins, cytolysins, and proteases (5) that may be harmful when
expressed in the cytoplasm of the pathogenic bacterium. Furthermore,
the autotransporters are relatively large molecules with a typical
domain structure. The passenger domain that is expressed in the
cytoplasm may fold into a conformation that is incompatible with
translocation through the Sec-translocon, even when the -barrel
domain is still being synthesized.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 31-20-4447175; Fax: 31-20-4446979; E-mail: luirink@bio.vu.nl.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M211630200
2 E. N. G. Houben, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: Hbp, hemoglobin protease; IMP, inner membrane protein; IMV, inverted membrane vesicle; SRP, signal recognition particle; DSS, 2,2-dimethyl-2-silapentanesulfonic acid; TF, trigger factor; TPS, two-partner secretion.
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