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INTRODUCTION |
Proteins that are destined to be translocated across or inserted
into the bacterial inner membrane
(IM)1 are targeted to
transport sites by multiple mechanisms. In Escherichia coli,
many secreted proteins are targeted to the IM by molecular chaperones
such as SecB, which keep them in a loosely folded, translocation-competent conformation (1, 2). The chaperone-based targeting pathways promote the translocation of fully synthesized proteins in vitro and probably also function in a
post-translational fashion at least to some extent in vivo
(3, 4). By contrast, recent studies have suggested that a variety of
inner membrane proteins (IMPs) are targeted to the membrane by an
essential ribonucleoprotein complex that is closely related to the
eukaryotic signal recognition particle (SRP) (5-7). In mammalian
cells, SRP is a complex composed of six polypeptides and a single RNA
that targets proteins to the secretory pathway in a strictly
co-translational fashion (reviewed in Ref. 8). The 54 kDa subunit of
SRP (SRP54) binds to signal sequences of nascent polypeptides and
guides ribosome-nascent chain complexes to transport sites in the
endoplasmic reticulum (ER) via an interaction with the membrane-bound
SRP receptor. Although the SRP found in E. coli and many
other bacterial species contains only a single protein (a homolog of
SRP54 called "Ffh") and a small RNA ("4.5 S RNA") (9), many
aspects of its function appear to be conserved, including
co-translational binding to substrates (10) and a specific interaction
with a homolog of the SRP receptor ("FtsY") (11).
All of the different targeting pathways appear to converge at the IM.
Both exported proteins and IMPs are transported by a common
translocation channel or "translocon" composed of a
phylogenetically conserved heterotrimer called the SecYEG complex,
which is closely related to the Sec61p complex found in the eukaryotic
ER (reviewed in Ref. 12). Bacteria differ from eukaryotes, however, in
that they have a unique peripheral membrane protein called SecA that interacts with SecY (13, 14) and that plays an essential role in
protein export (15, 16). SecA acts as a molecular motor that binds to
SecB-preprotein complexes in the cytoplasm (17) and then uses the
energy of ATP hydrolysis to catalyze post-translational translocation
across the cytoplasmic membrane (18). Following translocation, SecA is
released from the membrane (19). Relatively little is known about the
role of SecA in IMP insertion, and its role in transporting proteins
targeted by the SRP pathway is particularly unclear. The insertion of
some IMPs, but not others, has been proposed to be
SecA-dependent (e.g. Refs. 20-24), but the SecA dependence does not correlate well with SRP dependence. Moreover, in
all of the studies on IMP insertion, SecA activity has been blocked by
adding sodium azide, a weak inhibitor of the SecA ATPase, or by
shifting strains containing the temperature-sensitive
secA51ts allele to high temperature. Because ATP
binding and hydrolysis regulates the affinity of SecA for different
components of the system, azide treatment may interfere with the normal
cycling of SecA and may therefore inhibit IMP insertion by an indirect mechanism. Likewise, the SecA51(Ts) protein becomes trapped on the IM
above 33 °C (25) and may block IMP insertion by simply interfering
with the docking of ribosomes. Indeed recent studies strongly suggest
that the use of sodium azide and temperature-sensitive sec
alleles can produce misleading results (22, 26).
To circumvent the problems associated with conditional alleles and
inhibitors of SecA function, we used a novel approach to investigate
the role of SecA in the membrane insertion of SRP substrates. Initially
we tested for a genetic interaction between SRP and SecA using a
synthetic lethality assay. The results from these experiments raised
the possibility that SRP and SecA function in the same biochemical
pathway. To test this idea directly, we examined the effect of SecA
depletion on the membrane insertion of an SRP substrate, AcrB. In these
experiments we used a strain that contains a secAam
mutation and a temperature-sensitive amber suppressor. At low temperature full-length SecA protein is synthesized, but at high temperature only a small unstable fragment of the protein is produced (16). We found that after SecA depletion AcrB was quantitatively retained in the cytosol, where it was eventually degraded.
Surprisingly, we found that although SecA depletion also had a profound
effect on protein export, a fraction of at least one protein was still properly translocated. These results demonstrate that SecA function is
at least as important for the insertion of IMPs targeted by SRP as for
protein export and suggest that SecA plays a role in the transport of
both co-translationally and post-translationally targeted proteins.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Manipulations--
Strains HDB90
(secA+ zab::Tn10), HDB91
(secA450 zab::Tn10), and HDB92
(secA51ts zab::Tn10) were
constructed by introducing the secA mutations from MM52 and
EE450 (27, 28) into HDB45 (MC4100 pHDB4 ffh::kan) (6) by P1 transduction. Strains BA13 (MC4100 supFts
trpam secA13am
zch::Tn10) and DO251 (MC4100
supFts leu::Tn5
zch::Tn10) were obtained from Dr. Don Oliver. Media preparation and bacterial manipulations were performed according to
standard methods (28). Selective media contained 100 µg/ml ampicillin
or 40 µg/ml chloramphenicol.
Plasmid Construction--
Amino acids 266-1049 were deleted
from AcrB by excising an NruI-SalI fragment from
plasmid S215 (6) and repairing the ends with DNA polymerase (Klenow
fragment) to generate plasmid S215
2. To create a fusion of alkaline
phosphatase (AP) with AcrB at amino acid 265 (pJN4), S215
2 was
digested with SalI and ligated to a
BsiHKAI-XhoI fragment of pHI-1 containing the AP
gene (30) using the oligonucleotide adapters
5'-CCGCCGGGTGCAGTAATATCGCCT-3' and
5'-TCGAAGGCGATATTACTGCACCCGGCGGTGCT-3'.2
A derivative of pJN4 in which the AP fusion was subcloned into pACYC184
(pJN6) was used for the experiments described here. A pACYC184
derivative containing the AcrB 576-AP fusion (pNU88) and plasmid pAP-1
have been described (6, 31).
Pulse-Chase Labeling, Immunoprecipitations, and Western
Blots--
Cells were grown overnight at 30 °C in M9 medium
containing 0.4% glucose and 40 µg/ml L-amino acids,
excluding methionine and cysteine. Cultures were then diluted into
fresh medium at an optical density (A550) of
0.02 and grown to an OD of 0.05 at 30 °C. Each culture was then
divided in half. One-half was maintained at 30 °C while the other
was shifted to 41 °C. At various times after the temperature shift,
cells were subjected to pulse-chase labeling and processed essentially
as described (6). In some experiments, spheroplasts were divided into
two portions, one of which was treated with proteinase K. Proteins were
collected by trichloroacetic acid precipitation, and
immunoprecipitations were performed as described (6, 31). To provide an
internal standard, chloramphenicol acetyltransferase (CAT) was
immunoprecipitated from all samples. To measure levels of SecA and
SecY, portions of each culture were removed and cold trichloroacetic
acid was added to a final concentration of 10%. trichloroacetic acid
precipitates were collected by centrifugation, and Western blotting was
performed as described (6). The antibodies against various proteins
that were used in this study were obtained from 5 Prime
3 Prime, Inc., Boulder, CO (AP, CAT), Dr. Jon Beckwith (ribose-binding protein
(RBP), OmpA), Dr. Don Oliver (SecA) and Dr. Koreaki Ito (SecY).
Subcellular Fractionation--
Radiolabeled cells were
resuspended in phosphate-buffered saline and lysed by ultrasonic
treatment using a Heat Systems XL-2020 sonicator. A "total" cell
lysate was obtained after removing unbroken cells by centrifugation at
3,000 × g for 10 min in a Sorvall HS-4 rotor. Cell
envelopes were separated from the cytosol by centrifugation at
100,000 × g for 30 min at 4 °C in a Beckman Optima
TLX tabletop ultracentrifuge. To separate the outer and inner
membranes, the pellet was resuspended in 3 mM EDTA (pH 7.2)
and incubated in the presence of 0.5% Sarkosyl for 20 min at room
temperature (32). This mixture was recentrifuged at 100,000 × g for 30 min at 4 °C. The pellet, which contained outer
membranes, was resuspended in 50 mM Tris-HCl (pH 8.0), 10 mM EDTA. Proteins were collected by trichloroacetic acid
precipitation and immunoprecipitations were performed as described above.
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RESULTS |
SecA Mutations Are Lethal in Strains That Have SRP
Deficiencies--
Although cells can generally tolerate slight
deficiencies in essential biochemical pathways, it has often been
observed that combining two deficiencies in the same pathway has a
synergistic effect that leads to cell death (33). Thus, if SRP and SecA are required in successive steps of IMP insertion, then reduction in
the activity of each of these factors might produce similar physiological effects that together would be lethal. To test the effect
of reducing SRP and SecA activity simultaneously, we constructed strains HDB90 (secA+), HDB91
(secA450), and HDB92 (secA51ts) in
which the expression of ffh is regulated by the
trc promoter. Cells were plated on LB agar containing
various amounts of IPTG and incubated at temperatures that are
permissive for growth. Based on previous studies performed with the
parent strain (HDB45), it was expected that a near wild-type level of
Ffh would be produced in the presence of 10 µM IPTG (6).
Growth of HDB91 cells at 37 °C and of HDB92 cells at 30 °C was
observed on LB plates containing this concentration of inducer (Fig.
1, A and B). When
the level of inducer and therefore the level of Ffh was slightly
reduced, however, strong growth defects were observed; HDB91 and HDB92 did not grow at all on plates containing 2 µM IPTG and no
IPTG, respectively. HDB90 cells grew about as well on the plates
containing reduced levels of inducer as on plates containing 10 µM IPTG. These results demonstrate that reduction in SRP
concentration in cells that contain SecA mutations produces a synthetic
lethal effect and therefore raise the possibility that SecA
participates in the insertion of SRP substrates.

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Fig. 1.
Ffh deficiencies are lethal in
secA mutant strains. HDB90 (secA+),
HDB91 (secA450), and HDB92 (secA51ts)
cells, which contain the ffh gene under control of the
trc promoter, were streaked on LB agar containing the
indicated concentration of IPTG. At IPTG concentrations below 10 µM, the cells contain less Ffh than isogenic
ffh+ strains. Plates were incubated at 37 °C for 22 h (A) or 30 °C for 28 h (B).
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SecA Depletion Abolishes the Insertion of AcrB--
We next
used a protease protection assay to study the effect of SecA depletion
on the insertion of the SRP substrate AcrB. In this assay, protease
treatment releases the AP domain from IMP-AP fusion protein molecules
that are properly inserted into the IM but not from those that are
retained in the cytoplasm (34). This method has been used to show that
inhibition of the SRP pathway sharply reduces the membrane insertion of
both the polytopic AcrB 576-AP fusion and the bitopic AcrB 265-AP
fusion (6).2 Strains BA13 (secAam
supFts) and DO251
(secA+ supFts) were first
transformed with plasmids that encode the AP fusions. Cells were then
grown in defined minimal medium at 30 °C, at which temperature the
amber suppressor in BA13 is stable. Under these conditions the two
strains grew equally well (Fig.
2A, diamonds and
circles) and contained similar amounts of SecA (Fig.
2B, lanes 3-4) as shown by Western blot. When
the cells reached early log phase, half of each culture was removed and
incubated at 41 °C, a temperature at which the amber suppressor is
denatured (Fig. 2A, solid arrow). Effective
dilution of the residual SecA protein required 3 h (Fig.
2B, lane 1) at which point cell growth began to
decline (Fig. 2A, dashed arrow). Portions of each
culture were removed at various times after the temperature shift and
radiolabeled with [35S]methionine and
[35S]cysteine. The cells were harvested, spheroplasts
were generated and treated with protease, and the released AP plus any
protease-protected AP fusion protein that remained was
immunoprecipitated with anti-AP antibodies.

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Fig. 2.
Growth of a
secAam strain and
depletion of SecA at high temperature. Panel
A, BA13 (supF (Ts) secA13 (Am)) and DO251
(supF (Ts) secA+) cells were transformed with plasmid pJN6
or pNU88 and grown in minimal medium at 30 °C (see "Experimental
Procedures"). When the optical density (A550)
reached 0.05 (filled arrow), the cultures were divided and
half of the cells were placed at 41 °C. The growth of BA13
(  ) and DO251 ( - - ) maintained at 30 °C and BA13 ( - ) and DO251 ( ... ) after the
shift to 41 °C is shown. Panel B, the level of
SecA in BA13 cells growing at 41 °C (lane 1) or 30 °C
(lane 3) and DO251 cells growing at 41 °C (lane
2) or 30 °C (lane 4) was measured by Western blot
3 h after the cultures were divided (panel A,
dashed arrow). An equal amount of protein was loaded in each
lane.
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We found that depletion of SecA had a profound effect on the membrane
insertion of the AcrB-AP fusion proteins. A partial block of AcrB
insertion was observed by 1.5 h after the shift (data not shown),
but by 3 h a virtually complete block was observed. All of the
pulse-labeled AcrB 265-AP fusion protein in BA13 cells was
protease-protected following a 2-min chase, indicating that the protein
was retained in the cytoplasm (Fig.
3A, lanes 1-2). Even after a 30-min chase, little or no fusion protein was inserted into the membrane (Fig. 3A, lane 4). Because the
fusion protein that remained in the cytoplasm was slowly degraded, only
about 50% of the radiolabeled molecules were immunoprecipitated at
this time point. By contrast, the AcrB 265-AP fusion protein was
inserted efficiently into the IM of BA13 cells grown continuously at
30 °C and DO251 grown at either 30 or 41 °C (Fig. 3A,
lanes 5-16) as indicated by the complete susceptibility of
the AP domain to proteolysis at all time points. Similar results were
obtained in experiments in which the insertion of the AcrB 576-AP
fusion was examined. At 41 °C, only protease-protected pulse-labeled fusion protein was immunoprecipitated from BA13 cells following a 2-min
chase (Fig. 3A, lanes 1-2). After longer chase
times, very little fusion protein was isolated from either untreated or
protease-treated spheroplasts (Fig. 3B, lanes
1-2) due to rapid degradation of the protein in the cytoplasm.
Quantitative insertion of the AcrB 576-AP fusion was observed in BA13
cells grown at 30 °C and in DO251 cells at both high and low
temperature (Fig. 3A, lanes 5-16), and the
membrane-bound form of the protein was stable (Fig. 3B,
lanes 3-8).

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Fig. 3.
SecA depletion completely blocks the
insertion of AcrB-AP fusion proteins. BA13 and DO251 transformed
with plasmid pJN6 or pNU88 were grown as described in the legend to
Fig. 2. Panel A, the insertion of AcrB 265-AP or AcrB 576-AP
in cells grown at 41 °C for 3 h or maintained continuously at
30 °C was examined by pulse-chase analysis. AP-containing
polypeptides were immunoprecipitated from untreated spheroplasts
(lanes 1, 8, 9, and 16) or
from spheroplasts that were treated with proteinase K (lanes
2-6 and 10-15). The length of the chase is
indicated. Panel B, the stability of AcrB 576-AP was
measured by immunoprecipitating the fusion protein from untreated
spheroplasts generated in the experiment described in panel
A.
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To confirm that the protease resistance of the AcrB-AP fusion proteins
was due to retention in the cytoplasm, we examined their localization
in more detail by cell fractionation. BA13 and DO251 cells were grown
at 30 °C as described above and shifted to 41 °C for 3 h.
The cells were then pulse-labeled and harvested following a 10-min
chase. Cell extracts were prepared by sonication, and membranes were
isolated by high speed centrifugation. About 80-90% of the AcrB
265-AP fusion protein in BA13 cells was found in the high speed
supernatant and cofractionated with the cytoplasmic protein CAT (Fig.
4A). By contrast, almost all
of the fusion protein in DO251 cells was found in the membrane
fraction. Similar results were obtained when the cells were
spheroplasted and lysed by hypotonic shock using established methods
(data not shown). These results demonstrate directly that depletion of
SecA prevents the integration of SRP substrates into the IM.

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Fig. 4.
Intracellular localization of AcrB 265-AP and
OmpA after SecA depletion. BA13 and DO251 transformed with plasmid
pJN6 were grown at 30 °C as described in the legend to Fig. 2. When
the A550 reached 0.05, cultures were shifted to
41 °C and incubated for 3 h. Aliquots were pulse-labeled and
subjected to a 10-min chase. Cell fractions were obtained as described
under "Experimental Procedures." Panel A, AcrB 265-AP
and CAT were immunoprecipitated from the total cell extract
(T, lane 1), the cytoplasm (C, lane 2)
and total membranes (M, lane 3). Panel B, OmpA
and its precursor (pro-OmpA) were immunoprecipitated from the total
cell extract (T, lane 1), the cytoplasm (C, lane
2), inner membranes (IM, lane 3) and outer membranes
(OM, lane 4).
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SecA Depletion Inhibits but Does Not Completely Abolish Protein
Export--
Although the effect of inhibiting SecA activity on protein
export has been studied extensively in vivo, most
experiments have involved treating cells with sodium azide or shifting
strains containing the secA51ts allele to the
nonpermissive temperature. To test the effect of SecA depletion on
protein export, BA13 and DO251 cells were first transformed with a
plasmid expressing an AcrB-AP fusion or with plasmid pAP-1 (31). Cells
were then grown and radiolabeled as described above, and AP, RBP, and
OmpA were immunoprecipitated from samples that were not treated with
proteinase K. Consistent with previous studies on SecA function, the
export of AP and RBP was completely blocked by SecA depletion. None of
the radiolabeled precursor was converted to the mature form in BA13
cells grown at 41 °C even after a 30-min chase (Fig.
5, lanes 1-3). Like the AcrB-AP fusion proteins, pre-AP retained in the cytoplasm (but not
pre-RBP) was slowly degraded during the long chase period. By contrast,
rapid export of both AP and RBP from BA13 grown at 30 °C or from
DO251 cells grown at either high and low temperature was indicated by
the complete processing of the radiolabeled precursors within 2 min
(Fig. 5, lanes 4-12). Export of OmpA was also significantly inhibited by SecA depletion, but surprisingly a small fraction of the
protein appeared to be slowly translocated across the inner membrane
(Fig. 5, lanes 1-3). Only about 12% of the protein was processed after a 2-min chase, but after 30 min, more than 20% of the
pro-OmpA was converted to OmpA. The remainder of the pro-OmpA was
gradually degraded in the cytoplasm.

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Fig. 5.
Depletion of SecA blocks protein export.
BA13 and DO251 were transformed with pAP-1 or pJN6. Cells were grown as
described in the legend to Fig. 2. AP, RBP, and OmpA were
immunoprecipitated from radiolabeled BA13 and DO251 grown at 41 °C
for 3 h (lanes 1-6) or maintained at 30 °C
(lanes 7-12). The length of the chase is indicated.
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We next performed cell fractionation experiments to confirm that the
processing of OmpA observed after SecA depletion was due to proper
translocation and insertion of the protein into the outer membrane
(OM). Pulse-labeled BA13 and DO251 cells that had been incubated at
41 °C for 3 h were harvested after a 10-min chase. Cell
membranes were separated from the cytoplasm by centrifugation and then
further divided into IM and OM fractions. The AcrB-AP fusion protein
was used as a marker to validate the membrane fractionation method
(data not shown). OmpA was then immunoprecipitated from each cell
fraction. In BA13 cells, all of the processed OmpA was correctly
localized to the OM (Fig. 4B, lanes 1 and
4). Most of the pro-OmpA remained in the cytoplasm, although
a small portion was isolated in the IM fraction (Fig. 4B,
lanes 1-3). As expected, all of the OmpA in DO251 cells was
properly processed and inserted into the OM (Fig. 4B,
lanes 1 and 4). Taken together, these results show that OmpA is accurately transported across the IM (albeit with
reduced efficiency) even after a severe reduction in the level of SecA.
Thus the IMP insertion defects described above are not due to a
nonspecific inactivation of translocons following SecA depletion.
To determine whether SecA depletion affected translocon stability, we
measured the SecY levels in BA13 and DO251 cells grown at 30 °C and
41 °C by Western blot. A similar amount of SecY was present in each
strain at both temperatures (Fig. 6,
lanes 2-5). This result strongly suggests that the AcrB
insertion block observed in the absence of SecA was not caused by a
loss of protein transport capacity.

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Fig. 6.
Depletion of SecA does not destabilize the
SecY complex. BA13 and DO251 cells were grown as described in the
legend to Fig. 2. The intracellular level of SecY in an equal number of
DO251 and BA13 cells grown at 41 °C for 3 h (lanes
1-3) or maintained continuously at 30 °C
(lanes 4 and 5) was measured by Western blot. An
extract from DO251 cells that overproduce translocon proteins was
loaded in lane 1 to provide a marker for the position of
SecY (*).
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DISCUSSION |
In the experiments reported here we have obtained strong evidence
that SecA plays an essential role in the membrane insertion of proteins
targeted by SRP in E. coli. In an initial genetic test, we
found that slight Ffh deficiencies are lethal in SecA mutant strains.
This observation indicated that SecA and SRP are likely to function in
either the same or in parallel biochemical pathways. To distinguish
between these two possibilities, we analyzed the insertion of a model
SRP substrate, AcrB, in cells that had normal levels of SRP but reduced
levels of SecA activity. To avoid possible artifacts associated with
conditional alleles and metabolic inhibitors in these experiments, we
used a strain in which SecA can be depleted. The observation that the
insertion of both bitopic and polytopic AcrB-AP fusion proteins was
quantitatively blocked by SecA depletion strongly supports the
hypothesis that SecA is required for the insertion of SRP substrates.
In light of evidence that SRP targets proteins to the IM
cotranslationally, the simplest explanation of the cell fractionation
data is that the SecA dependence is dictated by the AcrB moeity of the
fusion proteins. If SecA were required only for the transport of the AP
domain, then the fusion proteins would have been expected to
fractionate with cell membranes. Nevertheless, we cannot completely
exclude the possibility that attachment of an AP domain to an IMP
influences the SecA-dependence of its biogenesis. Interestingly,
results from a recent biochemical study (35) are consistent with the
conclusion that SecA functions downstream from SRP in the IMP insertion
process. Nascent IMPs that were bound to E. coli SRP were
released by the addition of FtsY and then, in the presence of membrane
vesicles, transferred to the translocon. Although some of the nascent
chains were cross-linked to SecY, a much larger fraction was
cross-linked to SecA. Our results suggest that the proximity of the
nascent chain to SecA that is implied by this study may have an
important functional significance.
The finding that SecA is required for the insertion of SRP
substrates in E. coli is surprising in light of previous
studies on the eukaryotic SRP pathway. In mammalian cells a tight seal between the ribosome and the translocon is generally formed after nascent chains are targeted to the ER by SRP (36). Continued elongation
of the nascent chain is thought to be sufficient to push the
polypeptide through the translocon. If the molecular mechanisms of
translocation are conserved in bacteria, then motor proteins such as
SecA would not be expected to participate in the transport of SRP
substrates. Thus our results, together with the observation that only
the core components of the translocon are evolutionarily conserved,
suggest that there are significant differences between eukaryotic and
prokaryotic translocation systems. It is possible that the composition
of the translocon or of the IM itself hinders the formation of a tight
ribosome-translocon junction or necessitates the use of a molecular
motor to drive proteins across the membrane. Dissociation of the
ribosome from the translocon has been observed in mammalian cells under
some circumstances (37), and this phenomenon might be more predominant in bacterial cells. If so, then the motor function of SecA may be
required to ensure continuous transport of nascent chains across the membrane.
An alternative explanation of our results that would be consistent with
the formation of a tight ribosome-translocon junction is that SecA
promotes the insertion of SRP substrates by a mechanism that does not
depend on its motor activity. Extensive analysis of protein
translocation in vitro has clearly shown that SecA uses the
energy of ATP hydrolysis to facilitate post-translational translocation, but the data do not rule out the possibility that SecA
has additional functions. Recently it has been shown that the ER
lumenal protein BiP maintains the permeability barrier of the ER by
sealing nontranslocating Sec61p complexes (38). It is conceivable that
inactive translocons in bacteria are sealed by a different mechanism
(no BiP equivalent has yet been identified) and that SecA triggers
conformational changes that are required to initiate both protein
translocation and IMP insertion. The observation that SecY is stable
after substantial SecA depletion suggests that SecA is not required,
however, to maintain the structural integrity of the translocon.
In addition to providing evidence that SecA is required for the
insertion of SRP substrates, our experiments also yielded several
unanticipated results. Given that inhibition of the SRP pathway only
partially blocks the insertion of all substrates that have been tested
(5, 6, 26),2 the observation that SecA depletion completely
abolished the insertion of AcrB is striking. This result suggests that
the insertion of AcrB is SecA-dependent regardless of the
mechanism by which it is targeted to the translocon. Furthermore,
although our data are consistent with other types of evidence
indicating that SecA is essential for protein export, the slow
translocation of OmpA observed after SecA was nearly completely
depleted was very surprising. OmpA is often used as a model protein for
in vitro translocation studies, and its transport into
membrane vesicles is strictly dependent on SecA (16). The relative
insensitivity of OmpA export to reduced SecA concentrations suggests
that it has an atypical ability to persist in a transport-competent
form or that it has a much higher affinity for SecA than most other
periplasmic and membrane proteins. In either case, the unexpected
behavior of OmpA in these experiments, together with the finding that
SecA is required for the transport of a wider range of proteins than previously suspected, suggests that many aspects of SecA function remain to be elucidated.