(Received for publication, September 17, 1996, and in revised form, November 12, 1996)
From the Based on the finding that a series of engineered
proOmpAs containing disulfide-bridged loops of different sizes at
different positions exhibits a discontinuous mode of polypeptide
transit across the cytoplasmic membrane of Escherichia
coli, we suggested previously that the translocation of
preproteins takes place at every 30 amino acid residues (Uchida, K.,
Mori, H., and Mizushima, S. (1995) J. Biol. Chem. 270, 30862-30868). In the present study, we investigated the molecular
mechanism underlying this stepwise translocation. Deletion or
relocation of hydrophobic segments of the mature domain of proOmpA (H1,
residues 233-237; H2, residues 261-265) significantly altered the
pattern of the stepwise translocation. The stepwise mode of polypeptide
insertion was also observed with reconstituted proteoliposomes
comprising purified SecA, SecY, and SecE. Cross-linking experiments
involving a photoactivable cross-linker revealed that SecY and SecA are
the components which interact with the hydrophobic segment of proOmpA.
The present results indicate that the hydrophobic segments of the
mature domains of preproteins interact with membrane embedded
translocase during polypeptide transit across the membrane, which
causes a discontinuous mode of polypeptide movement.
The translocation of preproteins across the cytoplasmic membrane
of Escherichia coli is mediated through interactions of
molecules, including a set of Sec proteins (1-5). The driving force
for translocation is provided by the proton motive force and the energy formed through SecA-mediated hydrolysis of ATP (6-9). SecY, SecE, and
SecG, a heterotrimeric membrane-embedded translocase, provide an
intramembrane pathway for preprotein transit (10-12). Biochemical studies, such as purification of the components of the translocase and
their reconstitution into proteoliposomes of specified compositions in
E. coli systems, have been performed (13, 14). These studies revealed the minimum components required for translocation and their
modes of action in substantial detail. However, the details of the
mechanism by which preproteins move through the cytoplasmic membrane
remains to be elucidated.
In previous studies, we showed that a proOmpA derivative containing a
disulfide-bridged loop between Cys-290 and Cys-302 can be translocated
across everted membrane vesicles of the cytoplasmic membrane of
E. coli. In the absence of the proton motive force, on the
other hand, translocation ceased when the loop reached the membrane
(15, 16). Taking advantage of this phenomenon, we analyzed how
preproteins are translocated across the membrane. We changed the
position of one of the cysteine residues (cysteine-290) toward the N
terminus so as to obtain proOmpA derivatives with disulfide-bridged
loops of different sizes at different positions (17). We expected that
the lengths of the translocated polypeptides would become shorter as
the first cysteine residue becomes closer to the N terminus, if
translocation ceases at the position of the loop. In contrast to our
expectation, however, we found that the size of the translocated
polypeptides remained almost the same for OmpA derivatives containing
loops of 10-25 and 29-59 amino acid residues, respectively, with a
change of about 3 kDa. This suggested that the in vitro
translocation of proOmpA through the secretory machinery takes place in
every 30 amino acid residue segments. Under certain conditions, the
movement of a polypeptide chain by about 20 amino acid residues during
translocation was also demonstrated upon the addition of a
nonhydrolyzable ATP analog, ATP Although the insertion-deinsertion cycle of SecA may partly explain the
stepwise translocation of preproteins, the details of the mechanism
remained unclear. It is quite difficult to imagine that SecA strictly
recognizes every 30 amino acid residue segments, which have a wide
variety of physicochemical properties. We therefore assume that the
mature domains of preproteins have some elements that control their
stepwise movement.
In the present study, we examined whether or not hydrophobic segments
of the mature domain of proOmpA determine the stepwise movement during
translocation across everted membrane vesicles of E. coli.
We focused on the fact that many outer membrane proteins in addition to
OmpA periodically contain hydrophobic segments in their mature domains,
although they are hydrophilic in general (19). We relocated or replaced
hydrophobic segment(s) with an artificial hydrophilic sequence(s), and
compared the patterns of stepwise movement.
Everted membrane vesicles were isolated from
E. coli K003 (Lpp pTD-T7, an expression
vector carrying the T7 promoter, was a generous gift from Dr. Date
(24). 1.3-kilobase pair EcoRI-HindIII fragments
encoding mutant ompA genes were isolated from the pOA series
plasmids described by Uchida et al. (17) and then subcloned into the EcoRI-HindIII site of pTD-T7. The
resultant plasmids are referred to as pTDL16-L43. The pTDL plasmids
were transformed into E. coli CJ236, and uracil-containing
single-stranded phagemid DNA was isolated. Oligonucleotide-directed
mutagenesis was performed according to the method of Kunkel (25). The
following oligonucleotide primers were used: proOmpAL16- 43 Translated 35S-labeled proOmpA
derivatives were oxidized with ferricyanide and then subjected to
in vitro translocation essentially according to the method
of Uchida et al. (17). The translocation mixture (25 µl)
comprised a 35S-labeled proOmpA derivative, membrane
vesicles (5 µg of protein), 2 mM ATP (or 5 mM
AMP-PNP), 5 mM MgSO4, 50 mM
potassium phosphate (pH 7.5), 15 µg/ml SecB, and 40 µg/ml SecA.
Dithiothreitol was added to the final concentration of 5 mM, if necessary. The ATP concentration was maintained by
regeneration with 10 mM creatine phosphate and 10 µg/ml
creatine kinase. After 10-min incubation at 37 °C, the mixture was
treated with proteinase K (200 µg/ml) for 20 min at 0 °C,
subjected to trichloroacetic acid precipitation, and then analyzed by
SDS-polyacrylamide gel electrophoresis and fluorography. Translocation
of looped proOmpAs with reconstituted proteoliposomes was performed
according to the method of Akimaru et al. (14).
ProOmpAL43C302Q, in which a cysteine
residue was introduced at Cys-260 was replaced with a glutamine
residue, was also created. 35S-Labeled proOmpA was
conjugated with APDP via Cys-260 according to the method of Joly and
Wickner (26). Briefly, APDP was dissolved in dimethyl sulfoxide (3 mg
in 50 µl) and then diluted 200-fold with 100 mM potassium
phosphate (pH 7.5). To 70 µl of the 35S-labeled proOmpA
dissolved in 6 M urea and 100 mM potassium
phosphate (pH 7.5), 100 µl of diluted APDP was added. After 1 h
at room temperature, proteins were precipitated with trichloroacetic
acid, and then washed with acetone twice, then the washed
precipitate was dissolved in 6 M urea and 50 mM
potassium phosphate (pH 7.5).
After in
vitro translocation reaction and photolysis, samples were
solubilized in a solution comprising 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% SDS, diluted 33-fold with
dilution buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 100 µM 5,5 Purified SecA was iodinated according to the method of
Economou and Wickner (18) with a minor modification, as described below. Na125I and SecA were incubated on ice for 20 min in
a tube that had been coated with IODO-GEN. The reaction mixture was
transferred to another tube containing 2 mM dithiothreitol
and then applied to a Sephadex G-50 column to remove
Na125I. To prepare 125I-SecA-bound membrane
vesicles, 4 nM 125I-SecA and 200 µg/ml
urea-washed membrane vesicles were incubated for 15 min on ice in a
100-µl reaction buffer consisting of 50 mM Tris-HCl (pH
8.0), 50 mM KCl, 5 mM MgCl2 and 0.2 mg/ml bovine serum albumin. The membrane vesicles were isolated and
then resuspended in the same buffer. 125I-SecA-bound
membrane vesicles, 1 mM ATP, 50 µg/ml SecB, 5 mM phosphocreatine, creatine kinase (10 µg/ml), and 20 µg/ml looped proOmpA derivative were incubated in reaction buffer for
15 min at 37 °C, followed by proteinase K treatment (final, 0.1 mg/ml) for 15 min on ice. Proteins were precipitated with
trichloroacetic acid and then analyzed by SDS-polyacrylamide gel
electrophoresis and fluorography.
We first looked at the hydropathy profile of the wild-type
proOmpA (Fig. 1A). There are two major
hydrophobic segments, referred to as H1 (residues 233-237) and H2
(residues 261-265), respectively, close to the N-terminal side of two
cysteine residues (Cys-290 and Cys-302). Fig. 1B summarizes
the positions of the cysteine residues used for the formation of a
disulfide bridge with those of the two hydrophobic regions. We
constructed, by the oligonucleotide-directed mutagenesis method, genes
encoding derivatives of proOmpA in which either or both of the
hydrophobic segments were replaced with artificial hydrophilic amino
acid sequences (Fig. 1D). As judged from the hydropathy
profile, the replacement was expected to render both hydrophobic
segments completely hydrophilic (Fig. 1C).
A, hydropathy profile of proOmpA.
Hydropathy was calculated according to Kyte and Doolittle with a span
of seven residues. Hydrophobic stretches H1 and H2 are indicated by
bold bars. B, structures of proOmpA derivatives. The amino
acid residues of the signal peptide (
These proOmpA derivatives were then subjected to in
vitro translocation into everted membrane vesicles of E. coli in the absence of the proton motive force (Fig.
2). As shown in Fig. 2A, the sizes of
proteinase K-resistant fragments (band A) were essentially the same for proOmpAs containing loops of 16 and 21 amino acid residues
(L16 and L21). When the size of the loop increased (L35), a sudden
decrease in the size of the fragment, by about 3 kDa, occurred and
several fragments were observed (collectively referred to as band
B). The sizes of the fragments were the same for L35 and L43. L29
exhibited an intermediate profile between those of L21 and L35. These
results were essentially the same as those of Uchida et al.
(17). This stepwise profile was not due to the substrate specificity of
proteinase K, because similar profiles were observed when other
proteases with different substrate specificities were used (17).
As shown in Fig. 2B, when the H1 segment was replaced with a
hydrophilic sequence, the sizes of the protease K-resistant fragments were the same for all proOmpA derivatives (L16, L21, L29, L35, and
L43). Similar results were obtained when trypsin was used instead of
proteinase K (data not shown), suggesting that the observed profile was
not due to the specificity of the protease. When the H2 segment was
replaced with a hydrophilic one, no fragment was observed at the
position of band A for L16 or L21. Instead, bands
C* and C were observed for L16 and L21,
respectively. It should be noted that a slight decrease in size
occurred between L16 and L21 (Fig. 2C), suggesting that
translocation occurred continuously. When both the H1 and H2 segments
were replaced, the size of the proteinase K-resistant fragments
continuously decreased as the loop size increased, except for L35 and
L43 (Fig. 2D). Discontinuous translocation between L35 and
L43 can be explained by the fact that appropriate proteinase K cleavage
sites do not exist from around residues +260 to +252, where the
translocation of L43 is predicted to cease at the position of its loop.
These results suggest that the hydrophobic segments are involved in the
stepwise movement. Similar profiles were observed when translocation
was performed in the presence of the proton motive force (data not shown), suggesting that the mechanism of SecA-dependent
translocation is principally the same in the absence and presence of
the proton motive force, as we discussed previously (17). Furthermore, when the translocation reaction was conducted in the presence of
everted membrane vesicles derived from a strain harboring the mutation
in the prlA666, which was shown to be a particularly strong
suppressor for a variety of maltose-binding protein export defects
(27), the patterns of the translocation of looped proOmpA derivatives
were the same as that obtained in the presence of the wild-type
membranes (data not shown).
To confirm that the hydrophobicity is important for the stepwise
movement, we inserted another H1 segment just before the original H1
segment to increase the hydrophobicity (proOmpA2xH1). As shown in Fig.
2E, the hydrophobic segment 2xH1 showed substantial stop-translocation activity. The size of proteinase K resistant fragment, 26 kDa, was the same as that of the proOmpA containing loops of 43 amino acid residues (L43), implying that the
translocation of proOmpA2xH1 was interrupted at the H1 region. These
results suggest that the hydrophobicity itself is important for the
translocation stall.
If hydrophobic segments really determine the discontinuous transition
of proOmpA through the membrane, it was expected that the relocation of
hydrophobic segments would lead to a change in the stepwise
translocation pattern. We therefore constructed a series of proOmpAs,
in which hydrophobic segment H1 was shifted about 14 residues closer to
the N terminus, and then subjected them to in vitro
translocation (Fig. 3A). Proteinase K
digestion of the translocated mutant proOmpAs with the relocated H1
region (proOmpAH0+2) revealed a change in the stepwise profile (Fig. 3B). In the case of wild-type proOmpA derivatives, a shift
of band A to B occurred around a loop size of 29 (Fig. 2A),
whereas proOmpA derivatives with the relocated H1 region showed a shift at a loop size of 35. This result strongly supports the idea that hydrophobic segments are directly involved in the discontinuous transition of a polypeptide across membrane vesicles.
When the in vitro
translocation reaction was performed with a low concentration of ATP
(10 µM), translocation intermediates of proOmpA were
observed (7, 9). We examined whether or not the proteinase K-resistant
fragment that was formed on partial translocation of proOmpA with a
disulfide-bridged loop was identical with translocation intermediates
accumulated with a low concentration of ATP. A low concentration of ATP
permitted a slow translocation reaction and thereby resulted in
protease-resistant intermediates, termed I29 and
I16 (Fig. 4, lane 1).
Translocation intermediate I29 showed the same mobility as
band B on SDS-polyacrylamide gel electrophoresis, indicating that the
translocation was interrupted at the same site (Fig. 4, lanes
1 and 2). On the other hand, limited translocation of
the proOmpA devoid of the H1 and H2 segments gave several bands, which
gave a different pattern from I29 (Fig. 4, lane
3). These results suggest that hydrophobic segments in the mature
domain of proOmpA are responsible for the accumulation of translocation
intermediates with a low concentration of ATP.
Reconstitution studies revealed that SecA, SecE,
and SecY are the minimum components mediating preprotein translocation
(13, 14). To determine whether or not these components are enough for
the stepwise movement of proOmpA derivatives, an in vitro preprotein translocation reaction with reconstituted proteoliposomes comprising purified SecA, SecE, and SecY was carried out with a series
of looped proOmpAs. As shown in Fig. 5, proOmpAs with disulfide-bridged loops exhibited a discontinuous mode of
translocation, which was similar to that observed with everted membrane
vesicles. This implies that the stepwise movement of proOmpA
derivatives is achieved only through SecA, SecE, SecY, and
phospholipids.
Using a
photoactivable and reducible cross-linker, APDP, Joly and Wickner (26)
showed that OmpA in a translocation intermediate is cross-linked to
SecA and SecY, suggesting that translocation occurs through a
proteinaceous channel. To determine whether or not the H2 segment
interacts with SecA and SecY during translocation, we introduced a
cysteine residue (Cys-260) in the H2 segment and replaced Cys-302 with
a glutamine residue and then APDP was attached to the cysteine residue
of the radiolabeled proOmpA. The translocation reaction was conducted
with a low concentration of ATP (10 µM) to produce
translocation intermediates (Fig. 6A, lane
2). After membrane vesicles had been isolated by centrifugation to
remove free 35S-proOmpAL43C302Q, they were irradiated with
UV light. This photolysis allowed a reactive group attached to Cys-260
to form a covalent bond with its nearest neighbor. To determine whether
or not SecA and SecY are cross-linked, immunoprecipitation was
performed as described before (28) and then the precipitates were
analyzed by SDS-polyacrylamide gel electrophoresis in the presence of
dithiothreitol. Treatment of the precipitates with dithiothreitol
resulted in the release of 35S-proOmpAL43C302Q from the
attached nearest neighbor. Most of the cross-linked
35S-proOmpAL43C302Q was specifically precipitated by
antisera directed against SecY (lane 4), whereas a
part of the cross-linked 35S-proOmpAL43C302Q was
precipitated by antisera raised against SecA (lane 5). When
ATP was absent, no significant amount of
35S-proOmpAL43C302Q was immunoprecipitated by either
anti-SecY (lane 7) or anti-SecA (lane 8),
indicating that cross-linking with SecY and SecA occurred only when
partial translocation had taken place. After the photolysis, when
excess purified antigen (purified SecA and SecY) was added to the
reaction mixture, the amount of precipitated 35S-proOmpAL43C302Q was drastically reduced (Fig.
6B). Since SecA repeats the insertion-deinsertion cycle
during translocation, cross-linking of 35S-proOmpAL43C302Q
with SecA might occur during its insertion into the membrane. These
results suggest that the H2 segment interacts mainly with SecY, and
partly with membrane-embedded SecA, when translocation is arrested.
This is in good agreement with the idea of Joly and Wickner (26) that
the interaction of the translocating chain with translocase occurs
initially through SecA followed by SecY.
We next examined whether or not the presence of
hydrophobic segments in the mature domain of proOmpA affect the
insertion of SecA into the membrane. 125I-SecA was
incubated with ATP and proOmpA containing a disulfide-bridged loop to
form membrane-inserted SecA. Proteolysis of the membrane-inserted SecA
gave a 30-kDa fragment, as observed for the membrane-inserted SecA,
which had been formed with wild-type proOmpA (Fig. 7).
Removal of the H1 and H2 segments from the looped proOmpA did not alter the efficiency of membrane insertion of 125I-SecA,
suggesting that the hydrophobic segments play no role in the insertion
of SecA into the membrane.
The results of our previous study involving proOmpAs with
disulfide-bridged loops of different sizes at different positions suggested that the in vitro translocation of proOmpA through
the secretory machinery takes place in every 30 amino acid residues (17). How can such exact synchronization of the stepwise movement be
achieved from the N terminus to the C terminus of proOmpA? Although the
secretory machinery including SecA, which repeats the
insertion-deinsertion cycle during translocation (18, 29), may be
responsible for the stepwise movement of preproteins, preproteins themselves may also have elements that control such movement. In the
present work, we carried out systematic analysis of the stepwise
movement of proOmpAs with different distributions of hydrophobic
segments in the mature domain of proOmpA. Our results strongly indicate
that hydrophobic segments are determinants for the stepwise movement of
proOmpA. We also demonstrated that the formation of translocation
intermediates with a low concentration of ATP (7, 9) is due to the
hydrophobic segments in the mature domain of preproteins. Hydrophobic
portions in the mature domain may transiently be arrested in the Sec
machinery in the process of translocation.
The observation that proOmpA translocation takes place in about every
30 amino acid residue segments (17) can be explained by the fact that
the distance between the H1 and H2 segments is about 30 residues. We
assume that the loop and hydrophobic segments cooperatively act to
arrest the transit of a polypeptide chain across the membrane. When the
loop size is small, as in the cases of L16 and L21, the H1 segment
passes through the membrane, and the H2 segment acts as an arrest
signal (Fig. 1A). The H2 segment may be a stronger arrest
signal than the H1 segment because the arrest of proOmpA transit
occurred at the H2 segment, but not at the H1 segment, when the
concentration of ATP was low (Fig. 4). When the loop size is larger,
the H1 segment arrests the transit of a polypeptide, probably due to
steric hindrance or another reason. It should be noted that the
distance between the two hydrophobic segments is about 30 amino acid
residues. This coincides with the difference in size between bands A
and B. When the H1 segment is replaced with a hydrophilic sequence, the
H2 segment arrests the transit irrespective of the loop size. When the
H2 segment is replaced, translocation continuously occurs after the H1
segment has passed through the membrane (Fig. 2C). It was
expected that the replacement of both segments results in continuous
translocation. However, as shown in Fig. 2D, L35 and L43 did
not exhibit continuous translocation. The reason for this is not clear
at present. When the H1 segment is shifted toward the N terminus by 14 amino acid residues, the H2 segment is the dominant arrest signal even
if the loop size is as large as 35 amino acid residues (L35). In L35,
the H0 segment may pass through the membrane before the loop approaches
the membrane, therefore the H2 segment acts as an arrest signal. On the
other hand, in wild-type L35, the H1 segment may not have passed
through when the loop approaches the membrane.
Hydrophobic segments, which cause the stepwise translocation, consist
of four hydrophobic amino acid residues. Such sequences seem to be
appropriate for transfer through the membrane, i.e. the
hydrophobicities of such hydrophobic segments are below the threshold
of the hydrophobicity required for the stop of transfer. It is possible
that proOmpA translocation pauses not only at H1 and H2, but also at
other short hydrophobic segments in the mature domain.
Similar discontinuous translocation may occur in other outer membrane
proteins. Outer membrane proteins generally form cross- A polypeptide crosses the membrane through a tunnel or pore comprising
SecY, SecE, and SecG (26). On the other hand, the peripheral
cytoplasmic factor, SecA, plays roles in both initiation and the
elongation step of the entire process of the polypeptide transit (7,
9). In the process of translocation, a SecA arm accompanies the
polypeptide chain into the membrane-embedded translocase, so that a
portion of SecA is transiently located in the membrane, as revealed by
the occurrence of a 30-kDa SecA fragment that is resistant to
proteolytic cleavage (18). We showed here that the hydrophobic segments
in the mature domain of proOmpA derivatives had no effect on the
formation of the 30-kDa proteolytic fragment of SecA. This may suggest
that SecA does not participate in the specific recognition of
hydrophobic segments and that other proteins such as SecY may play this
role. Further study is required to clarify this possibility.
In conclusion, our present observations clearly showed that short
segments comprising four hydrophobic amino acid residues are
determinants for the stepwise translocation. Since such segments are
ubiquitous in secretory proteins (19), stepwise movement may be common
for protein translocation across the E. coli cytoplasmic membrane.
We thank Dr. Philip J. Bassford, Jr. of the
University of North Carolina for the donation of strain JP136 and Mie
Okazaki for her skillful technical assistance.
Research Laboratory of Resources
Utilization,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
S1 or
AMP-PNP (7). SecA may be involved in this discontinuous polypeptide
transition. The binding of ATP to SecA results in the membrane
insertion of a 30-kDa fragment of the SecA protein (18), which may be
accompanied by the insertion of segments of preproteins into the
membrane.
Materials
,
uncB-C-Tn10)
as described previously (20). SecA was purified as described previously
(21). Mutant proOmpAs were purified as described by Crooke et
al. (22), and SecB was purified as described by Weiss et
al. (23). All proOmpA derivatives used for the in vitro
translocation reactions were synthesized in vitro in the
presence of EXPRE35S35S protein labeling mix
(DuPont NEN) (17). SecE and SecY were purified as described by Akimaru
et al. (14). Na125I (100 mCi/ml) was purchased
from ICN. IODO-GEN, 5,5
-dithiobis-(2-nitrobenzoic acid), and
N-[4-(p-azidosalicylamido)butyl]-3
-(2
-pyridyldithio)propionamide (APDP) were from Pierce. Irradiation of samples was performed with a UV
lightbox from Funakoshi (Funa-UV-linker FS-1500). ATP, creatine kinase,
and creatine phosphate were obtained from Boehringer Mannheim.
Proteinase K was purchased from Merck. Restriction enzymes were from
Takara Shuzo Co. Sephadex G-50 (medium) and protein A-Sepharose CL-4B
were purchased from Pharmacia Biotech Inc.
H1,
5
-GTCGGTGTAACCCGTACGCTCATCTTTACCGT-3
(splI);
proOmpAL16-29
H2,
5
-GCCGGGATACCTTTGGACGTACGCTCATCTTTAACAGACTGAGCACGG-3
(splI); proOmpAL35
H2,
5
-GCCGGGATGCATTTGGACGTACGCTCATCTTTAACAGACTGAGCACGG-3
(splI); proOmpAL43
H2,
5
-GCCGGGATACCTTTGGACGTACGCTCATCTTTACATGTCTGAGCACGG-3
(splI); proOmpAL16-29
H1+2,
5
-GGGATACCTTTGGAGAGCTCTTTATCACGAACAGACTGAGCACGG-3
(SacI); proOmpAL35
H1+2,
5
-GGGATGCATTTGGAGAGCTCTTTATCACGAACAGACTGAGCACGG-3
(SacI); proOmpAL43
H1+2,
5
-GGGATACCTTTGGAGAGCTCTTTATCACGACATGTCTGAGCACGG-3
(SacI); proOmpAL16-43
H0,
5
-CAAGTTGCTCAGAACAACTACGAGCTGATCCAGAG-3
(
); proOmpAL43C302Q,
5
-CGATCCGGAGCCAGCTGGTCGATCAGTG-3
(PvuII);
proOmpA2xH1, 5
-AGCGTGGTCGTGTTGTGCCGAGTTGTTCTGGGTTAC-3
(BamHI) and 5
-CGATCCGGAGCCAGCTGGTCGATCAGTG-3
(PvuII); proOmpA2xH1C, 5
-GTAACCCAGAACAACTGCGCACAACACGACCAC-3
(FspI). The
restriction sites created are shown in parentheses and the deleted site
is underlined. Using these primers, the complementary DNA strand was
synthesized and the resulting double-stranded DNA was transformed into
JM109. To obtain double-stranded DNA for the
H1+2 series, uracil-containing single-stranded phagemid DNA was isolated from the
H1 series, and then the complementary strand was synthesized. These
plasmids encoding various mutated ompA genes were subjected to in vitro transcription with T7 RNA polymerase, followed
by in vitro translation as described (15).
-dithiobis(2-nitrobenzoic acid), and 0.1% Triton X-100 and then mixed with an appropriate antibody, anti-SecY or anti-SecA. After incubation at 4 °C for 1.5 h, the antigen-antibody complexes were isolated with protein A-Sepharose CL-4B. The resin was washed with dilution buffer and then
incubated in sample buffer for electrophoresis at 37 °C for 5 min to
recover the bound proteins.
Construction of ProOmpA Derivatives Containing Disulfide-bridged
Loops
Fig. 1.
21 to
1) and the mature domain
(+1 to +325) are indicated with the positions of cysteine residues. The
amino acid residues replaced by a cysteine residue are shown in
parentheses. The name of each proOmpA derivatives, loop
size, and hydrophobic stretches H1 and H2 are also shown.
WT, wild-type. C, amino acid sequences of the H1
and H2 regions. Each segment was replaced by an artificial amino acid
sequence, which is shown with the amino acid residue positions. The
hydropathy patterns of these mutated proOmpAs are also shown.
D, structures of mutated proOmpA derivatives. The name of
each proOmpA with removed hydrophobic segment(s) is indicated.
[View Larger Version of this Image (17K GIF file)]
Fig. 2.
A-D, alignment of translocation
intermediates obtained oined on proteinase K treatment in the process
of translocation of the series of looped proOmpAs, with or without
removed H1 and/or H2 portion(s). Various 35S-proOmpAs with
a disulfide-bridged loop (L43-L16), in 8 M urea and 50 mM potassium phosphate (pH 7.5), were diluted in an assay mixture (25 µl) comprising 5 µg of everted membrane vesicles, 1 µg of SecA, 0.375 µg of SecB, 5 mM MgSO4,
and 2 mM ATP in 50 mM potassium phosphate (pH
7.5). After 10-min incubation at 37 °C, the samples were treated
with 5 µl of proteinase K (5 µg/µl) for 20 min on ice. The
samples were then recovered by trichloroacetic acid precipitation,
washed with acetone, and then subjected to SDS-PAGE and fluorography.
The positions of the translocation intermediates of OmpAs are indicated
by A and B. Models of the translocation
intermediates are also shown. E, structure of proOmpA2xH1 and its in vitro translocation. 35S-ProOmpA
derivatives were subjected to in vitro translocation as
described above. The position of the mature OmpA is indicated.
[View Larger Version of this Image (19K GIF file)]
Fig. 3.
Structure of proOmpAH0+2 and alignment of its
translocation intermediates obtained on proteinase K treatment.
A, comparison of the primary structures of wild-type
(WT) and proOmpAH0+2 (H0+2) with the amino
acid positions. B, the conditions for limited translocation and digestion with proteinase K were the same as those given in the
legend to Fig. 2. The positions of the translocation intermediates of
the OmpAH0+2 series are indicated by A and
B.
[View Larger Version of this Image (18K GIF file)]
Fig. 4.
Intermediates of various proOmpA derivatives
during translocation. The indicated 35S-proOmpA
derivatives were preincubated in assay mixtures under the conditions
given in the legend to Fig. 2, except for ATP, for 2.5 min at 37 °C.
The samples were further incubated for 10 min with 10 µM
ATP (lanes 1 and 3) or 2 mM ATP
(lanes 2 and 4), followed by proteinase K
treatment (final, 200 µg/ml) for 20 min at 0 °C, and then analyzed
by SDS-PAGE and fluorography. Translocation intermediates
I29 and I16 are indicated.
[View Larger Version of this Image (22K GIF file)]
Fig. 5.
Alignment of translocation intermediate bands
obtained on proteinase K treatment with reconstituted
proteoliposomes. Samples (15 µl) of reconstituted
proteoliposomes were mixed with 1.5 µg of SecA and 5 µl of 10 mM potassium phosphate (pH 7.5), containing 10 mM MgSO4, 10 mM ATP, 150 mM NaCl, and an ATP-generating system composed of 50 mM creatine phosphate and creatine kinase (1.25 mg/ml).
After 3-min preincubation at 37 °C, the reaction was started by the
addition of 35S-proOmpA. The translocated portion, which
became proteinase K-resistant, was detected by SDS-PAGE, followed by
fluorography. The positions of the translocation intermediates of OmpAs
are indicated by A and B. WT,
wild-type proOmpA with no disulfide-bridged loop.
[View Larger Version of this Image (11K GIF file)]
Fig. 6.
A, limited translocation and nearest
neighbor analysis. Translocation intermediates were prepared
under the conditions given in the legend to Fig. 4 with APDP-conjugated
proOmpAL43C302Q prepared as described under "Experimental
Procedures." APDP-proOmpAL43C302Q in urea was diluted in the
reaction mixture (25 µl) described in the legend to Fig. 2, without
ATP, followed by incubation at 37 °C for 2.5 min. After the 10-min
incubation with 10 µM ATP (lanes 2, 4, and
6) or with 5 mM AMP-PNP (lanes 3 and
5) at 37 °C, membrane vesicles were isolated by
centrifugation and then resuspended in buffer comprising 50 mM potassium phosphate (pH 7.5) and 5 mM
MgSO4. The samples were treated with proteinase K
(ProK) (200 µg/ml), shown in lane 2. The
samples were irradiated with an inverted lightbox at 256 nm for 6 min
at room temperature. The irradiated samples were then dissolved in
buffer comprising 50 mM potassium phosphate (pH 7.5), 5 mM MgSO4, and 1% SDS (total 25 µl), vortexed for 5 min, and then diluted with buffer consisting of 50 mM
Tris-Cl (pH 8.0), 150 mM NaCl, and 0.1% Triton X-100 to
33-fold. The diluted samples were then subjected to immunoprecipitation
as described under "Experimental Procedures" and then analyzed by
SDS-PAGE with a reducing agent. Lane 1 contains 10% of the
samples used in lanes 2-6. B, immunocompetition analysis.
Experiments were performed as described above in the presence of
purified SecA and SecY (lane 2, 50 µg each; lane
3, 5 µg each) and then precipitated with both anti-SecA and
anti-SecY antibody. Lane 4 contains 10% of the samples used
in lanes 1-3.
[View Larger Version of this Image (22K GIF file)]
Fig. 7.
SecA membrane insertion of looped proOmpA
derivatives. The reaction mixtures comprised TL buffer without
dithiothreitol (50 mM Tris-Cl (pH 8.0), 50 mM
KCl, 5 mM MgCl2, and 0.2 mg/ml bovine serum
albumin), 125I-SecA-bound membrane vesicles (200 µg/ml),
5 mM phosphocreatine and creatine kinase (10 µg/ml), as
described under "Experimental Procedures." For the reactions in
lanes 2-4 mutant proOmpA (20 µg/ml), ATP (1 mM), or both were added as indicated. The samples were
incubated at 37 °C for 15 min and then digested with proteinase K
(0.1 mg/ml) for 15 min on ice. After trichloroacetic acid
precipitation, the samples were analyzed by SDS-PAGE and fluorography.
The arrowheads indicate the protease-inaccessible 30-kDa
bands.
[View Larger Version of this Image (13K GIF file)]
-structures (30, 31), and their most hydrophobic regions are significantly less
hydrophobic than the membrane-spanning sequences of inner membrane
proteins (19). The lack of long hydrophobic regions in outer membrane
proteins is quite reasonable, because such regions hinder the transfer
of preproteins through the Sec machinery. On the other hand, inner
membrane proteins that span the bilayer one time have hydrophobic
regions of 20-25 amino acid residues. The lengths of these regions in
the
-helical conformation are long enough to span the membrane
(32).
*
This work was supported by grants from the Ministry of
Education, Science, Sports and Culture of Japan and by a Japan Society for the Promotion of Science for Young Scientists (to K. S.). 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.
§
Supported by a Research Fellowships of the Japanese Society for the
Promotion of Science for Young Scientists. To whom correspondence should be addressed. Tel.: 81-426-76-7116; Fax: 81-426-76-8866.
1
The abbreviations used are: ATPS, adenosine
5
-O-(thiotriphosphate); AMP-PNP, adenosine
5
-(
,
-imino)triphosphate; APDP, N-[4-(p-azidosalicylamido)butyl]-3
-(2
-pyridyldithio)propionamide; PAGE, polyacrylamide gel electrophoresis.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.