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INTRODUCTION |
The majority of the proteins destined for the extracytoplasmic
environment in Escherichia coli are synthesized as
precursors in the cytoplasm that are translocated across the inner
IM1 in a non-native state. To
reach their final conformation and destiny they need to be prevented
from premature folding and degradation in the cytoplasm. It is well
established that molecular chaperones and folding catalysts are
required to channel proteins into productive export, folding, and
assembly pathways. In the cytoplasm one of the first events that takes
place when a nascent PhoE emerges from the ribosome is the interaction
with trigger factor, a cytoplasmic chaperone. Using a cross-linking
approach, the latter protein was found to interact with nascent PhoE
chains as short as 57 amino acids (1-3). Subsequently, at a late
co-translational or early post-translational stage the precursor
protein interacts with SecB (4-6). This SecB·precursor
complex is then targeted to SecA, the peripheral subunit of the
membrane-embedded SecYEG complex (7). PhoE precursors are then
translocated across the IM in a non-native state. When appearing at the
periplasmic side of the IM, these polypeptides undergo complex folding
processes and have to be prevented from misfolding, aggregating, and
degrading. Proteins destined for the outer membrane or the
extracellular environment require correct targeting to these locations.
Periplasmic chaperones and folding catalysts have been implicated to be
involved in the folding and assembly pathway of OMPs. It was
demonstrated previously that OMPs are exposed to the periplasmic
chaperones during the assembly process (8). A periplasmic system
consisting of molecular chaperones and folding catalysts has been
described (9, 10). Among them, the peptidyl-prolyl isomerases (FkpA, SurA, RotA, and PpiD) carry out the isomerization around the Xaa-Pro peptide bond. In addition to the isomerase activity the FkpA and SurA
proteins were found to posses chaperone activity (11, 12). The second
type of folding catalysts are the protein disulfide isomerases (Dsb
proteins) carrying out disulfide bond formation and thiol-disulfide
exchanges (13).
In addition, Skp functions as a molecular chaperone in the periplasm of
Gram-negative bacteria, possibly specifically required for the
biogenesis of OMPs. Skp is a basic soluble protein in the periplasm,
but it was also found in IM fractions (14, 15). The protein exists in
two different states as was demonstrated by their different sensitivity
toward proteases (16). The conversion between these states can be
modulated in vitro by phospholipids, lipopolysaccharides,
and bivalent cations (16). The function of these two different forms of
Skp in vivo is not yet known. Previously, it was
demonstrated that Skp binds selectively to OmpA and proteins of the
bacterial porin family (16, 17). In addition, non-OMPs seem to act as a
substrate for binding (18). Skp interacts with OmpA in close vicinity
of the IM, and it was found to be involved in the release of the OMP
from this membrane (15). In addition, cells defective in Skp and the
periplasmic protease DegP accumulate protein aggregates in the
periplasm (15). These results suggest that Skp is a molecular chaperone
involved in generating and maintaining solubility of early folding
intermediates of OMPs in the periplasm.
The molecular mechanism of the PhoE assembly pathway has been
investigated extensively in vivo and in vitro.
However, little is known about which periplasmic chaperones and folding
catalysts are involved in the late stages of translocation over the IM, in the release from the IM, and in the early stages of folding in the
periplasm. Furthermore, the order of interactions with accessory
proteins and the timing of the various reactions are largely unknown.
In the studies described in this report, we used an unbiased approach
to study these processes. We present evidence that the initial
interaction of the non-native porin protein PhoE at the periplasmic
side of the IM is with Skp. This interaction occurs already early
during translocation of PhoE, i.e. when PhoE is still in a
transmembrane orientation in the translocase. At the N terminus of
PhoE, two Skp-binding sites could be identified. In contrast to OmpA,
release of PhoE from the IM appeared not to be dependent on Skp.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Growth Conditions--
The E. coli K12 strain MC4100 (F
lacU169
araD136 rpsL thi relA) was used for the
spheroplast labeling experiments and for the isolation of lysate and
inner membrane vesicles (19). MC4100 and RC354c, containing a deletion
in the skp gene (17), were used for the isolation of
periplasmic extracts (20). BG87 (21) was used for the isolation of
trigger factor-depleted lysate as described before (2, 19). Top10F'
(Invitrogen, Carlsbad, CA) was used for routine cloning procedures.
Strains were routinely grown in LB medium and the appropriate
antibiotics. For pulse labeling experiments a GB1 minimal medium was
used (22) supplemented with 0.4% maltose as the carbon source and an
18-amino acid mixture (0.1 mM) (without Met and
Cys).
Construction of Plasmids--
Plasmid pNN100 containing
the phoE gene under control of the tac promoter was
constructed previously (23). Plasmids pC4Meth300PhoE, pC4Meth100PhoE
ss, and pC4Meth137PhoE
ss were obtained by
polymerase chain reaction using pVG01 (24) as a template essentially as described in a previous study (2). The latter two plasmids contain
instead of a signal sequence a Met and a Ser residue just as in
pJP370 (25). Construction of plasmid pAra300PhoE.DHFR was as follows:
pC4Meth300PhoE was digested with BamHI and
HindIII and ligated to the
BamHI-HindIII fragment of pGEM4.DHFR containing the mouse dhfr gene (a gift from T. Langer, München,
Germany). Subsequently, the phoE-dhfr fusion gene was cloned
downstream of the arabinose promoter on plasmid pMPMK4 (26). Plasmid
pGAH317, carrying the wild-type skp gene, has been described
(27).
Spheroplast Labeling, Cross-linking, and Release--
For pulse
labeling of spheroplasts cells were grown in GB1 medium with casamino
acids during 7 h and then diluted for overnight growth in such a
way that the culture turbidity reached a value of ~0.5 (at 660 nm)
the next morning. 1.5 ml of cells was collected at 10,000 × g for 2 min, washed once with phosphate-buffered saline (0.15 M sodium chloride, 0.1 M sodium
phosphate, pH 7.0) and resuspended in 250 µl of 100 mM
Tris-HCl (pH 8.0). After addition of 250 µl of 100 mM
Tris-HCl (pH 8.0), 1 M sucrose, the mixture was incubated for 10 min at 0 °C. After addition of EDTA (4 mM) and
lysozyme (50 µg/ml), 500 µl of cold H2O was added
immediately. Cells were incubated on ice for 15 min, and the formation
of spheroplasts was followed by phase contrast microscopy.
MgCl2 was added to a final concentration of 20 mM to stabilize the spheroplasts, which were subsequently
collected at 10,000 × g for 2 min and resuspended in
0.5 ml of spheroplast labeling medium (GB1 medium containing 250 mM sucrose, 10 mM MgCl2). After
10-min incubation at 30 °C the spheroplasts were induced for the
synthesis of PhoE by adding 1 mM IPTG or of PhoE-DHFR by
adding 0.1% arabinose. After 5-min incubation with IPTG or 10 min with
arabinose, the spheroplasts were pulsed for 1 min with 16.5 µCi of
[35S]methionine (Amersham Pharmacia Biotech), followed by
a 5-min chase with 2 mM cold methionine. During the chase
50 µl of periplasmic proteins (~1 mg/ml) were added when indicated,
and in the case of a cross-link experiment also 1 mM
bis(sulfosuccinimidyl) suberate (BS3) dissolved in labeling
medium (Pierce). After 10 min the cross-linking reaction was quenched
on ice with quench buffer (0.1 M glycine, 10 mM
NaHCO3, 250 mM sucrose, pH 8.5). The
spheroplasts were subsequently collected by centrifugation for 2 min at
10,000 × g. Pellet and supernatant were either
trichloroacetic acid-precipitated and immunoprecipitated or used for
flotation gradient centrifugation. In release experiments the samples
were not treated with a cross-linker. The pellet and supernatant
fractions were trichloroacetic acid-precipitated and either directly
loaded (1/15 of the total material) on SDS-polyacrylamide gel
electrophoresis (PhoE) or immunoprecipitated (OmpA). The release values
for PhoE presented in the text are an average of at least three experiments.
Pulse-chase Labeling--
Cells were grown exactly as described
above and pulsed for 30 s with [35S]methionine (30 µCi per ml of cells) followed by a chase with cold methionine (final
concentration, 2 mM). Samples were taken at several time
intervals and precipitated with trichloroacetic acid.
Flotation Gradient Centrifugation--
To purify membranes and
membrane-associated protein complexes; crude membranes were subjected
to flotation gradient analysis as described previously (28).
Pellet fractions from the spheroplast labeling experiment were
resuspended in spheroplast-labeling medium and subsequently osmotically
lysed by dilution with 9 volumes of cold water. The lysed cells were
sonicated three times for 10 s. Non-lysed cells were
removed by low speed centrifugation (6000 × g for 2 min). Subsequently, membranes were pelleted by centrifugation in a
TLA120.2 rotor for 30 min at 300,000 × g and loaded
onto the bottom of a tube that was overlaid with sucrose. After
centrifugation, the two top fractions, containing the membranes and
membrane-associated proteins, were trichloroacetic
acid-precipitated.
In Vitro Transcription and Translation--
Ribosome nascent
chain complexes (RNCs) were prepared as follows. pC4MethPhoE-derivative
plasmids were linearized with HindIII and transcribed using
a T7 polymerase MEGAscript kit of Ambion Inc. (Austin, TX).
Translations were carried out as described previously (1) with the
following modifications. A lysate was used from a trigger
factor-depleted strain, and 100 µM aurin tricarboxylic acid was added 2.5 min after the start of the translation to inhibit initiation. RNCs were purified by centrifugation over a sucrose cushion
(29). Per standard translation reaction of 12.5 µl the RNCs were
resuspended in 2 µl of RN buffer (100 mM KOAc, 5 mM Mg(OAc)2, 50 mM Hepes-KOH, pH
7.9), mixed with 10.5 µl of periplasmic extract (~1 mg/ml), and
treated with 1 mM BS3. In vitro
translation of prePhoE was carried out as described previously (16).
Either plasmid pJP29 (30) (Fig. 7) or pVG01 (24) (Fig. 3) was used. For
targeting to the IM, vesicles were added at the start of the
translation reaction, and cross-linking was carried out with 1 mM DSS as described (1). Translation reactions were
transferred to ice and incubated for 5 min with chloramphenicol (30 µg/ml) to stop further translations. Subsequently, mixtures were
incubated with proteinase K (0.2 mg/ml) in the absence or presence of
Triton X-100 (1% w/v). The protease reaction was stopped after 30-min
incubation on ice by the addition of 1 mM phenylmethylsulfonyl fluoride.
Immunoprecipitations and Protein Analysis--
Trichloroacetic
acid pellets were either directly solubilized with SDS-sample buffer
for SDS-polyacrylamide gel electrophoresis-precipitated proteins or
solubilized in SDS-buffer and used in immunoprecipitation reactions
under denaturing conditions. Immunoprecipitations were carried
out as described previously (31). All samples were analyzed on 10 or
15% SDS-polyacrylamide gels. Co-immunoprecipitations of in
vitro synthesized PhoE proteins with antibodies directed against
Skp were performed as described previously (16). Radiolabeled proteins
were visualized by phosphorimaging using a Molecular Dynamics
PhosphorImager 473 and analyzed using the ImageQuaNT software from
Molecular Dynamics.
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RESULTS |
Skp Interacts with PhoE at the Periplasmic Side of the Cytoplasmic
Membrane--
PhoE is translocated over the IM in a non-native state.
Upon entering the periplasm, the protein has to be released from this membrane to assemble into the outer membrane in its native trimeric structure. We wanted to determine which chaperones and folding catalysts assist in these processes and to study the timing of the
interaction with these accessory proteins. To that end, we used an
unbiased approach to analyze the molecular environment of the newly
translocated PhoE protein or PhoE translocation intermediates at the
periplasmic side of the IM. MC4100 cells containing an inducible
phoE gene were converted into spheroplasts, induced for the
expression of PhoE, pulse-labeled with [35S]methionine,
and chased with cold methionine. Subsequently, cells were treated with
the membrane-impermeable cross-linker BS3 either in the
presence or absence of added periplasmic extract. After
immunoprecipitating the PhoE proteins in the pellet or supernatant fractions with anti-sera against PhoE, proteins that interact with this
OMP were identified. As shown in Fig. 1,
lanes 13 and 14, a cross-linking product with a
molecular mass of ~52 kDa was found in the supernatant fraction. The
52-kDa product corresponds in size to the combination of one molecule
of PhoE (36 kDa) and one Skp (16 kDa). The band was absent when
periplasmic proteins were not added (lane 11) or when a
periplasmic extract of a skp mutant was used (lane
12). These data indicated that Skp closely interacts with PhoE
during or after the process of secretion into the spheroplast medium,
as was shown previously (16).

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Fig. 1.
PhoE interacts with Skp in close proximity of
the cytoplasmic membrane. E. coli cells of wild type
MC4100 containing plasmid pNN100 were converted to spheroplasts,
induced with IPTG, and pulse-labeled. During the chase either buffer
(A) or a periplasmic extract was added from an
skp mutant (B), wild type (C), or an
Skp-overproducing strain (D). In addition, during the chase
the cross-linker BS3 was added (lanes 5-9,
11-14). The spheroplasts were collected by centrifugation,
and the pellet and membrane fractions were used in immunoprecipitation
experiments with either PhoE or Skp. The cross-link product of
PhoE with Skp is indicated with an arrow.
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In the spheroplast pellet fraction cross-linking products of ~71, 57, and 52 kDa were detected (lanes 5-8), suggesting that these
products are still associated to the IM. The intensity of the 71- and
57-kDa bands was independent of the addition of periplasmic proteins.
This indicates that the cross-linking adducts were not periplasmic, but
membrane-associated proteins. The cross-linking adducts were not
further identified, but the size of the 71-kDa product suggests that
this represents a PhoE dimer. The band of 52 kDa could be
immunoprecipitated with a specific antiserum raised against Skp
(lane 9). A faint reaction was also obtained without the
addition of a periplasmic extract containing Skp (lanes 5 and 6), indicating that the residual amount of Skp
associated with the spheroplasts interacted with PhoE. These results
showed that Skp interacts with PhoE at the level of the IM. It is,
however, also conceivable that the Skp-PhoE cross-linking product in
the pellet fraction represented an interaction of Skp with PhoE
aggregates. To distinguish between these possibilities, spheroplasts
were lysed and IMs were purified by flotation gradient centrifugation (Fig. 2). The vast majority of PhoE
protein was associated with the membranes present in fraction 2 (see
lanes 3, 4, and 6). In this fraction
the IM protein YidC (32) was detected as a control protein (results not
shown). Furthermore, the typical three cross-linking products of 72, 57, and 52 kDa were also detected in this membrane fraction. The 52-kDa
band could be immunoprecipitated with either
PhoE (lane
6) or
Skp (lane 8). The three cross-linking bands could also be obtained when the cross-linking was carried out after the
flotation (lane 4). These results showed that the Skp·PhoE interaction occurs in close proximity of the membrane, resulting in a
membrane-associated PhoE·Skp complex.

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Fig. 2.
The PhoE·Skp complex is
membrane-associated. E. coli cells of wild type MC4100
containing plasmid pNN100 were converted to spheroplasts, induced with
IPTG, and pulse-labeled. During the chase periplasmic extract was added
from an Skp-overproducing strain together with the cross-linker
BS3 (lanes 5-8). Membranes were isolated and
loaded on a flotation gradient. The gradient was fractionated into four
fractions, and the upper two fractions are shown. The fractions were
used in immunoprecipitation experiments with either PhoE
(lanes 5, 6) or Skp (lanes 7,
8). Lanes 1-4 contain the proteins that are
present in membrane fractions that were cross-linked after the
flotation. An asterisk indicate the cross-link product of
PhoE with Skp.
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The interaction of Skp with PhoE at the membrane was also investigated
in vitro. The precursor of PhoE was synthesized in an
E. coli-derived translation system in the presence of
inverted IM vesicles. The precursors could be imported into these
vesicles with an efficiency of ~25% as was monitored by their
resistance against externally added proteinase K (Fig.
3, lanes 1, 4, and 7). This efficiency of targeting and insertion was also
found in other studies with in vitro synthesized prePhoE
(33). Cross-linking with the membrane-permeable cross-linker DSS gave
rise to a cross-linking product of 52 kDa that could be
immunoprecipitated with anti-serum directed against PhoE (lanes
2, 5) and Skp (lanes 3 and 6).
This cross-linking product must have been formed, at least for a large part, inside the vesicles (periplasmic side), because it was resistant to externally added proteinase K (lanes 5 and 6).
Importantly, this proteinase K-resistant cross-linking product could
not be obtained when vesicles were used isolated from a skp
mutant (lanes 13 and 14). The 52-kDa
cross-linking product that could be immunoprecipitated with anti-Skp
(lane 11) was degraded by the protease (lane 14), indicating that the cross-linking adduct origins from the lysate, which
was isolated from the wild type.

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Fig. 3.
In vitro synthesized PhoE
binds to Skp at the periplasmic side of the inner membrane.
PrePhoE was synthesized in vitro using an S135 extract in
the presence of inner membrane vesicles isolated from wild type
(lanes 1-8) or from an skp mutant
(lanes 9-16). After translocation, the samples were treated
with the membrane permeable cross-linker DSS and either directly
immunoprecipitated or after treatment with proteinase K. An
asterisk indicates the cross-link product of PhoE
with Skp. Molecular masses are indicated in kDa.
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Skp Interacts with a Translocation Intermediate of PhoE--
We
subsequently wanted to determine whether Skp interacts with PhoE during
translocation across the IM. Previously, it was demonstrated that a
PhoE-LacZ hybrid protein spans the IM, with the PhoE moiety in the
periplasm and the
-galactosidase moiety in the cytoplasm. In
addition, such LacZ fusion proteins were shown to block translocation
of other proteins across the IM, due to jamming of the Sec pathway
(34). In other studies dihydrofolate reductase (DHFR) was used to
create OmpA-DHFR translocation intermediates that have similar effects
as LacZ fusions (35) (36). We made use of this strategy to study the
environment of PhoE translocation intermediates. We constructed a
hybrid protein containing the N-terminal 300 amino acid residues of
PhoE fused to mouse DHFR under control of the inducible arabinose
promoter. Upon induction with arabinose, cells harboring the
phoE300-dhfr gene fusion synthesized the hybrid protein and
stopped growing (results not shown). In a pulse-chase experiment it
could be demonstrated that under non-inducing conditions relatively low
amounts of the hybrid protein were synthesized (Fig.
4a). In these cells, the OmpA
protein was rapidly processed to the mature form (Fig. 4c).
Upon induction with arabinose the PhoE-DHFR hybrid protein was
synthesized to increased amounts and both the hybrid precursor as well
as the mature form were present even after 30 min (Fig. 4b).
In addition, the synthesis of OmpA was reduced and proOmpA accumulated,
indicating that it could not be translocated across the IM (Fig.
4d). These results indicated that the synthesis of the
PhoE-DHFR hybrid protein causes jamming of the Sec translocase.
Proteinase K treatment of spheroplasts that were induced for synthesis
of the hybrid protein and subsequently pulse labeled, showed that the
PhoE moiety of the mature hybrid protein was degraded by the protease,
whereas the DHFR moiety was not (results not shown). These data
demonstrate that the PhoE-DHFR hybrid protein is in a transmembrane
orientation, because it is accessible for protease added from the
outside and it blocks the translocation apparatus. The vast majority of
the translocons must be occupied with this fusion protein, because an
efficient block in protein export is observed.

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Fig. 4.
300PhoE-DHFR forms a
transmembrane translocation intermediate. The translocation of
300PhoE-DHFR and OmpA was studied in a pulse-chase experiment. MC4100
containing para300PhoE-DHFR was incubated with (b,
d) or without (a, c) arabinose. The
cells were immunoprecipitated with either PhoE (a,
b) or OmpA (c, d).
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To analyze the environment of these translocation intermediates,
spheroplasts were induced with arabinose, pulse-labeled in the presence
of additional periplasmic proteins, and subsequently treated with the
soluble cross-linker BS3. In the pellet fractions several
cross-linked bands could be identified after immunoprecipitation with
antisera raised against DHFR (Fig. 5,
lane 3). The strong cross-linking product of ~97 kDa
possibly represented a dimer of the hybrid protein. The very weak band
of ~65 kDa could also be immunoprecipitated with antiserum raised
against Skp (lane 2). In addition, by using sequential immunoprecipitation we found that this cross-linking product could be
immunoprecipitated first by anti-Skp and subsequently by anti-PhoE (lane 1). The 65-kDa cross-linking product was
membrane-associated, because it was found in the same two fractions of
a flotation gradient as the inner membrane protein YidC (32) (results
not shown). These results demonstrated that the PhoE-DHFR hybrid
protein interacted with Skp and that the interaction of Skp with PhoE occurred during translocation, i.e. when PhoE is in a
transmembrane orientation. The amount of PhoE-DHFR·Skp complex found
in this experimental setup is much less compared with the amount of
PhoE·Skp found in the experiments described above and shown in Fig.
1. It is possible that cross-linking targets are not properly exposed in the hybrid protein, but it is also conceivable that a C-terminal PhoE-truncate binds less efficiently to Skp compared with the full-length protein.

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Fig. 5.
The 300PhoE-DHFR translocation intermediate
interacts with Skp. MC4100 containing pAra300PhoE-DHFR was
converted to spheroplasts, induced with arabinose, and pulse-labeled.
During the chase, periplasm from an Skp-overproducing strain and the
cross-linker BS3 was added (lanes 1-3). Pellet
fractions were used in immunoprecipitation experiments with DHFR
(lane 3), Skp (lane 2), or Skp followed by
PhoE (lane 1). The cross-link product of 300PhoE-DHFR
with Skp is indicated with an arrow.
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The Skp Binding Site(s) in PhoE--
The results obtained so far
indicate an early interaction between Skp and the first 300 amino acids
of PhoE protein. To determine which regions of the PhoE protein are
important for Skp binding, we investigated whether Skp is able to bind
to shorter, nascent chains of PhoE. PhoE ribosome nascent chain
complexes (RNCs) were synthesized in vitro, isolated by
centrifugation over a sucrose cushion, incubated with a periplasmic
extract, and subsequently treated with BS3. In the past we
found that nascent chains as long as 50 amino acids could interact with
trigger factor (2). Because trigger factor will block binding sites on
the nascent chain in these experiments, it is required to use a trigger
factor-depleted lysate. In addition we used constructs that lack a
signal sequence to mimic the situation at the periplasmic side of the
membrane as good as possible. As shown in Fig.
6a, an RNC containing 137 amino acids of PhoE gave rise to cross-linking products of ~32 and
~48 kDa (lane 6). Both cross-linking products could be
immunoprecipitated with
Skp (lane 7) and represent most
probably complexes between the 137-mer of PhoE and monomeric and
oligomeric forms of Skp. The cross-linking products were not detected
when the isolated RNC complexes were incubated with control buffer
(lane 2-4). Similar results were obtained with a shorter
version of the PhoE protein. RNCs of a 100-mer of PhoE resulted in
cross-linking products of 29 and 45 kDa that could be
immunoprecipitated with
Skp (Fig. 6b, lanes 5,
6). These products were found when a wild type periplasm was
used but were absent when an extract was used of a skp
mutant strain (lanes 2, 3). The 100-mer of PhoE
exposed ~65 amino acid residues out of the ribosome, indicating that
a binding site for Skp is present at the extreme N terminus of
PhoE.

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Fig. 6.
Skp interacts with ribosome nascent chain
complexes of PhoE. a, ribosome nascent chain complexes
of 137 amino acids of PhoE were synthesized, purified over a sucrose
cushion, incubated with buffer (A) (lanes 1-4)
or a periplasmic extract of an Skp-overproducing strain (D)
(lanes 5-8), and subsequently cross-linked with
BS3. b, a 100-amino acid nascent chain was used
and either incubated with a periplasmic extract of an skp
mutant (B) (lanes 1-3) or of a wild type strain
(C) (lanes 4-6). Cross-linked products were
identified by immunoprecipitation with either PhoE or Skp. The
cross-link product of PhoE with Skp is indicated with an
arrow.
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Interaction of Skp with Mutant PhoE Proteins Synthesized in
Vitro--
De Cock et al. (16) demonstrated that Skp
interacts with PhoE proteins that were synthesized in vitro,
because these PhoE proteins were specifically precipitated with
antibodies directed against Skp. The absence or presence of the signal
sequence did not interfere with complex formation. We used this
approach to study the Skp-binding site of PhoE in more detail by using
a set of PhoE mutants with internal deletions in the mature part of the
protein. Wild type and mutant PhoE proteins were synthesized in an
E. coli derived in vitro system (Fig.
7, TL). After synthesis, purified Skp was added followed by the addition of puromycin to release
all nascent chains. PhoE proteins associated to Skp were immunoprecipitated with antibodies directed against Skp. In all cases
PhoE·Skp complexes could be precipitated (Fig. 7,
Skp). However, the relative amount of PhoE
that could be co-precipitated varied between the different mutants
(Table I). Deletion of amino acids 3-11
in the mature portion of PhoE protein reduced Skp binding efficiency
only moderately by ~35% as compared with full-length PhoE protein.
However, larger deletions in the N-terminal part of PhoE protein
reduced the binding efficiencies much further, i.e. >90%.
A deletion of amino acids 48-146 reduced the efficiency of binding
another 20-fold to >99%. This can be explained by assuming that two
independent Skp-binding sites exist in the N-terminal part of PhoE
protein, i.e. between 3-91 and 110-174 PhoE. Deletion of
amino acids 48-146 would delete a major part of both sites resulting
in an almost complete reduction of binding to Skp.

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Fig. 7.
Immunoprecipitations of in vitro
formed complexes between Skp and wild type or mutant PhoE
proteins with Skp antiserum. Wild type and mutant PhoE proteins
were synthesized as described under "Experimental Procedures." Part
of translations mixtures (TL) were analyzed directly (5 µl) demonstrating the position of all translation products (indicated
by a small arrow) and chloramphenicol acetyltransferase
(Cat). The other part (45 µl) was analyzed after
immunoprecipitations with Skp antiserum
( -Skp). Lanes: 1, mPhoE;
2, 3-11; 3, 3-18; 4, 3-42;
5, 8-91; 6, 110-174; 7,
249-298; 8, 63-287; 9, 204-330;
10, 56-93; 11, 48-146.
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Table I
Relative efficiency of immunoprecipitations of PhoE proteins with Skp
Deleted areas in the mutant proteins are indicated by numbers,
i.e. the deletion extends from amino acid in position
X to the Yth amino acid in the mature part. The
efficiency of immunoprecipitation of wild type (WT) is set at 100%
(amount precipitated with Skp anti-serum as a fraction of full-length
PhoE protein synthesized: on average 17% ± 3%). The efficiency of
immunoprecipitation of the various mutant proteins is expressed as a
fraction of that amount.
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A mutant PhoE protein containing the N-terminal 204 amino acids, but
lacking the C-terminal part (Fig. 7, lane 9) leads to a
reduction of the binding efficiency with ~65%, suggesting that indeed the N-terminal part of PhoE protein contains the most important Skp-binding sites. Interestingly, however, a smaller deletion in the
C-terminal part of PhoE, amino acids 249-298 (Fig. 7, lane 7), reduced the efficiency much further (>90%). In conclusion, the N-terminal region seems to be important for Skp binding whereas different parts of the C terminus of PhoE seem to influence the binding
to these N-terminal sites. However, it cannot be excluded that
differences in folding characteristics of the various deletion mutants
affect Skp binding due to alterations in exposure of the Skp-binding sites.
Skp Is Not Responsible for the Release of PhoE from the Inner
Membrane--
Recently, it was proposed that Skp is required for the
release of OmpA protein from spheroplasts. Furthermore, Skp was
proposed to be involved in the acquirement of a soluble periplasmic
form of newly translocated OMPs (15). From the data presented in Fig. 1
(lanes 1 and 15) we observed that PhoE remained
associated to the membrane to a large extent. Addition of a periplasmic
extract isolated from a Skp-overproducing strain (Fig. 1, lanes
4 and 18) did not significantly influence the amount of
released PhoE. To investigate the release of PhoE from the spheroplasts
we carried out release experiments and found that 55% of PhoE remained
associated to the membrane in a wild type strain. It has been reported
that the amount of Skp in spheroplasts is lower in the presence of bivalent cations (14). When we leave out magnesium ions from our
spheroplast preparations, the amount of PhoE that remained associated
to the membranes was slightly increased (63%). In these spheroplast
preparations the release of OmpA into the supernatant was 90%. On the
contrary, in the strain lacking Skp protein, PhoE and OmpA were both
for a large part detected in the pellet fraction (65 and 61%,
respectively). The data indicate that Skp is required for the release
and formation of a soluble form of OmpA protein, whereas the release of
PhoE seems to be independent of Skp.
 |
DISCUSSION |
Proteins destined for the extracytoplasmic environment pass the IM
in a non-native conformation. Upon entering the periplasm, these
proteins have to be protected immediately against degradation, aggregation, and misfolding. To that end, periplasmic chaperones are
supposed to be involved in directing these proteins into a productive
folding and/or assembly pathway. Little is known which periplasmic
chaperones and folding catalysts are involved in the late stages of IM
translocation and the early stages of folding. We used an unbiased
approach to study the early interactions in the periplasm of accessory
proteins with the OMP PhoE, and we found an early interaction with the
periplasmic chaperone Skp. It has been suggested that Skp plays an
important role in these early events as a specific chaperone for OMPs
(15, 16), but the timing and the location of this interaction was not
clear. The data presented in this paper showed that the initial
interaction of PhoE with Skp occurs at the periplasmic side of the IM.
A hybrid protein consisting of 300 N-terminal amino acids of PhoE
protein fused to mouse DHFR formed a transmembrane translocation
intermediate. This hybrid protein interacted with Skp as monitored by
protein cross-linking, indicating that PhoE interacts with the
chaperone already during translocation. In addition, the data implicate the presence of one or more Skp-binding sites in the N-terminal region
of PhoE. Indeed, we found that Skp was able to closely interact with an
N-terminal PhoE fragment of 65 amino acid residues in in
vitro cross-linking experiments. Furthermore, deletions in the
N-terminal as well as the C-terminal part of the PhoE protein resulted
in reduction of precipitation of PhoE with
Skp. The data can be
explained by assuming the presence of two Skp-binding regions in the N
terminus of PhoE. Absence of certain parts of the C terminus in PhoE
mutant proteins might influence the conformation of this protein, and,
as a consequence, the exposure of binding sites is different. The
immunoprecipitation experiments were performed in the absence of
membranes, which might influence the manner and extend to which Skp
interacts with PhoE protein. However, it should be emphasized that
detergents are present during immunoprecipitations, which can be
regarded as a membrane-mimicking environment. Taken together, the
results demonstrated that Skp interacts with PhoE protein once the
extreme N-terminal part, containing these binding sites, appears in the
periplasm. We therefore suggested that early translocation
intermediates of OMPs are already recognized by this molecular
chaperone. It is unlikely that the specific interaction of Skp with
OMPs is due to recognition of a linear amino acid sequence, because the
extreme N terminus of PhoE protein does not reveal a conserved
sequence. It is therefore more likely that the basis for the selective
interaction of Skp with OMPs is at the level of recognizing certain
structural elements, possibly
-strands or
-sheets.
Previously, it was demonstrated that Skp exists in two different states
characterized by their different sensitivities to proteases in
vivo (16). In addition, Skp was localized as a soluble periplasmic
as well as a membrane-associated protein. Both forms have apparently
the ability to interact with OMPs. In co-immunoprecipitation and
cross-linking experiments to ribosome-nascent chains, the soluble,
protease-sensitive form was able to form a soluble complex with OMPs.
Here, we demonstrated that full-length PhoE protein, imported into IM
vesicles, interacts with Skp that is co-purified with IM vesicles, most
likely due to association to the membrane. Similar results have been
reported previously for OmpA (15). Interestingly, we were not able to
detect an interaction of membrane-associated Skp with the PhoE-DHFR
hybrid. We had to add additional periplasmic extract, containing
soluble Skp, to obtain cross-linking. In contrast, cross-linking of Skp to full-length PhoE protein is possible without the addition of additional periplasmic proteins but the efficiency of cross-linking does increase when periplasm is added. These results do not necessarily mean that the membrane-associated form of Skp is not able to interact with the N-terminal portion of the PhoE protein in the fusion protein
per se. It might be that this part has no cross-linking targets in close proximity of Skp, but it is also possible that it is
has a reduced affinity for binding to membrane-associated Skp, thereby
decreasing the possibility for detection. In addition, it is possible
that the protease-sensitive Skp that was added with the periplasmic
extract is converted to the resistant form in the spheroplast
preparations. This would raise the Skp concentration to higher levels
at the membrane and allow detection of interaction with the fusion
protein by cross-linking. We propose that the initial interaction of
PhoE translocation intermediates occurs with the membrane-associated
form of Skp.
Schäfer et al. (15) suggested that Skp is involved in
generating and maintaining the solubility of early folding
intermediates of OMPs in the periplasm. In contrast to OmpA, however,
release of the PhoE protein is not dependent on Skp. PhoE remains
associated to the membrane to a large extent even in wild type
spheroplasts. The release of PhoE from the spheroplasts was not
significantly influenced by bivalent cations or the addition of
addition of periplasmic proteins. Possibly, de novo
synthesis of another or an additional component is necessary to get
release of PhoE or the PhoE·Skp complex from the cytoplasmic
membrane. It is not clear why the porin protein PhoE behaves
differently as compared with OmpA in this respect. One possibility is
that the assembly of the trimeric porins requires a different assembly
route as compared with the monomeric protein OmpA. In this context it
is interesting to mention that assembly of porins but not OmpA requires de novo synthesis of lipidic components (either
lipopolysaccharide and/or phospholipids) (37-40). Possibly, the
observed differences in the release of OmpA versus that of
the PhoE protein from spheroplasts is a reflection of this difference
in the assembly route.