(Received for publication, May 5, 1995; and in revised form, January 29, 1996)
From the
We examined the dependence of protein export and membrane protein insertion on SecE and SecA, two components of the secretion (Sec) apparatus of Escherichia coli. The magnitude of the secretion defect observed for signal sequence-containing proteins in cells depleted of SecE is larger and more general than that in many temperature- or cold-sensitive Sec mutants. In addition, we show that the proper insertion of the polytopic MalF protein (synthesized without a signal sequence) into the cytoplasmic membrane is also SecE-dependent. In contrast to an earlier study (McGovern, K., and Beckwith, J.(1991) J. Biol. Chem. 266, 20870-20876), the membrane insertion of MalF also is inhibited by treatment of cells with sodium azide, a potent inhibitor of SecA. Therefore, our data strongly suggest that the cytoplasmic membrane insertion of MalF is dependent on the same cellular machinery as is involved in the export of signal sequence-containing proteins. We propose that the mechanism of export from the cytoplasm is related for both signal sequence-containing and cytoplasmic membrane proteins, but hydrophobic membrane proteins such as MalF may have a higher affinity for the Sec apparatus.
Translocation of proteins across
the cytoplasmic membrane in Escherichia coli(2,
3) is catalyzed by a machinery whose components have
been identified both genetically and biochemically. These components
include the SecA, SecE, and SecY proteins, which are essential for protein
export, and at least four other proteins (SecB, SecD, SecF, and SecG) that
greatly enhance protein translocation, especially at lower temperatures.
Proteins localized by the Sec ()apparatus to the periplasm or outer membrane are
synthesized with an N-terminal signal sequence that is proteolytically
cleaved from the mature portion of the protein during export by the action
of leader peptidase.
In vivo studies of the dependence of a particular protein on the Sec machinery for proper cellular localization have relied largely on the use of conditional Sec missense mutants. These mutant strains accumulate untranslocated, signal sequence-bearing proteins in their cytoplasm under nonpermissive conditions. The extent of a secretion defect in cells can thus be directly assessed by examining the amount of precursor species versus mature species of a given secreted protein. The analysis of the Sec dependence of protein export is made complicated by the fact that different proteins are not equally sensitive to the export inhibition conferred by conditional sec mutations. For example, in a strain deleted for the secDF genes, ribose-binding protein and maltose-binding protein (MBP) are exported relatively poorly(4) . In these same strains, OmpA is exported fairly well (the majority of protein synthesized in a short pulse is exported by 10 min), and DegP is exported with wild type kinetics. It is possible that OmpA and DegP have a greater affinity for the secretion apparatus than do ribose-binding protein or MBP.
The assessment of whether a protein is dependent upon the secretion apparatus for translocation is further confounded by the nature of the sec conditional mutations. These mutations, rather than causing the synthesis of a conditionally defective Sec protein, often result in lower levels of functional Sec protein and, in effect, reduce the quantity of functional secretion apparatus in the cell. This phenomenon has been thoroughly characterized for the secE cold-sensitive mutations(5) . The translocation of proteins that have a higher affinity for this limited secretion apparatus would be less affected relative to those proteins that have a lower affinity. Recently, a more accurate assessment of the Sec dependence of proteins has been made possible by the development of strains in which individual Sec proteins can be conditionally depleted(4, 6) . These strains contain either the secDF genes or the secE gene under control of the highly repressible araBAD promoter. Strains in which expression of the secDF genes is repressed have a very severe and general secretion defect for signal sequence-containing proteins after depletion of the SecD and F proteins(4) . In this study, we characterize the secretion defect of cells that have been depleted for the SecE protein.
Integral cytoplasmic membrane proteins often have large hydrophilic domains that must be translocated to the extracytoplasmic face of the membrane. However, most cytoplasmic membrane proteins are synthesized without cleavable N-terminal signal sequences. Whether most of these polypeptides depend on the Sec machinery for translocation of hydrophilic domains through the hydrophobic membrane is unresolved. A few cytoplasmic membrane proteins have been tested for their dependence on the Sec machinery for membrane insertion, including M13 procoat protein, leader peptidase (Lep), the methyl-accepting chemotaxis receptor protein Tsr, the ProW component of the ProU osmoregulatory system, and the MalF component of the maltose transport complex(1, 7, 8, 9) . These analyses have suggested that the M13 procoat, ProW, and MalF are Sec-independent, whereas Lep and Tsr are Sec-dependent.
Recently, an highly sensitive assay for the proper
insertion of the MalF protein into the cytoplasmic membrane and its
subsequent assembly into the hetero-oligomeric maltose transport complex
was described (10) . This assay is based on the
accessibility of the periplasmic domains of MalF (3, 180, 30, and 30
residues, respectively; (11) ) to exogenous
proteases. The sensitivity of this assay in combination with the new SecE
depletion mutant led us to readdress the dependence of MalF on the Sec
apparatus for the export of its periplasmic regions.
The MalF protein in all
experiments is expressed at physiological levels from the normal
chromosomal copy of the gene in the malB locus at 92 min on the
E. coli chromosome; therefore there are no artifacts from protein
overproduction. Strains BT62, BT8, and BT10 are malT derivatives
of MC4100 (F
,
lacU169, thiA, relA, rpsL, araD139). BT8 and BT10 are
Mal
and
malE
malF
malG
malK
, respectively (described in (10) ). BT62 is malE
malF
malG
malK
with secE
19-111, pcnB80 zadL::(Tn10 Tc
Str
),
recA::cat, and pCM22.
BT7 is Mal P1 transductant of KM1089, a
malT
,
malF derivative of the MC1000 strain DHB3
(araD139,
(ara-leu) 7697,
lac
X74,
phoA PvuII,
phoR, thi, rpsL; 1). BT63 is azi-4 Leu
P1 transductant of BT7.
Cultures of CM124 were grown with aeration at 37 °C in M63 minimal medium supplemented with 0.4% D-glucose or L-arabinose, 0.2% glycerol, thiamine, and all amino acids except cysteine and methionine. Cultures of BT62 for proteolysis experiments were grown in the same fashion except with either 0.2% glucose or arabinose.
To deplete BT62 of SecE, cultures were diluted 1:100 from
an overnight culture grown with arabinose into fresh M63 + glycerol +
arabinose. After 2-4 h of growth, the arabinose-supplemented culture was
split. One portion of the culture was maintained in medium + glycerol +
arabinose. The cells from the other portion of the culture were washed in
M63 medium + glycerol and then resuspended in M63 + glycerol + 0.2%
glucose. Cultures were maintained at A of 0.2-0.4 during SecE depletion and
S labeling.
Cells were labeled for 30 s with 40-50 µCi/ml [S]methionine using Expre
S
S label (New England Nuclear).
For proteolysis experiments with strain BT62, cells were induced with 1 mM
cAMP (Sigma) for 5 min before the addition of radiolabel to amplify the
expression of the mal genes. When NaN
was used, it
was added to 2 mM (during the cAMP induction for strain BT62) for 3 min
before labeling. After labeling, cold methionine was added to 0.05%, and
incubation at 37 °C continued for 10 min. Afterwards, the pulse and
chase samples were chilled to 0 °C, harvested, and converted to
spheroplasts as described previously(12) .
Proteolysis was done on 0.5-ml portions of labeled cells for 20 min at 0
°C with 25 µg/ml freshly prepared trypsin (Worthington Enzymes)
and stopped by the addition of phenylmethylsulfonyl fluoride (Sigma) at
0.4 mg/ml and soybean trypsin inhibitor (Boehringer Mannhiem) at 25
µg/ml. After labeling/proteolysis, cells were harvested and
resuspended in 50 µl of 50 mM Tris, pH 7.6, at 25 °C, 2% SDS, 1
mM EDTA (TSE) and heated to 65 °C for 20 min. Control
immunoprecipitations of MBP were done from 100-µl portions of labeled
cells that were resuspended in 50 µl of TSE and heated to 65 °C
for 20 min. TSE samples each were diluted into 0.8 ml of 50 mM Tris, pH
8.0, 150 mM NaCl, 2% Triton X-100, 1 mM EDTA buffer, and proteins were
immunoprecipitated as described with appropriate antibodies and
Staphylococcal protein A (12) . Samples were analyzed
by SDS-PAGE on 12.5% resolving gels. Quantitation of proteins was done
with a Molecular Dynamics PhosphorImager(12) , and
the data presented represent the averages of three experiments. Proteins
were detected in Western blot analysis as described(10) , using a goat anti-rabbit conjugated secondary antibody
(Boehringer Mannhiem).
Figure 3: The effect of SecE depletion
and NaN-treatment on
MalF protease sensitivity. Strains BT62 (MalF
) and KM1089 (
MalF) were grown and
treated as described under ``Materials and Methods.'' Sec
samples were grown in
permissive conditions (in glycerol, no NaN
for KM1089, in
glycerol + arabinose, no NaN
for BT62).
SecE
BT62
samples were grown in glucose for 3.5 h. SecA
samples were grown in permissive
conditions, and NaN
was added
before labeling. A, pulse-labeled (P) or pulse-labeled and
chased (C) samples were untreated(-) or treated (+) with
trypsin prior to immunoprecipitation with MalF antiserum. The radiolabeled
intact MalF protein and three MalF tryptic peptides (produced under
Sec
conditions:
F1, F2, and F3) are visualized here after SDS-PAGE
and fluorography with quantitation provided in Table 1. Background bands unrelated to MalF but recognized
by the antiserum are shown (a, b, c, and d).
B, a portion of the TSE-solublized samples from the proteolysis
experiment shown were analyzed after SDS-PAGE by Western blotting with
MalK antiserum. Pulse samples without trypsin are in lanes 1 and
3 for KM1089 and lanes 5, 8, and 12 for BT62.
Pulse samples with trypsin are in lanes 2 and 4 for KM1089
and lanes 6, 9, and 13 for BT62. Chase samples
without trypsin for BT62 are in lanes 10 and 14 (and data
not shown for BT62 + glucose at 3.5 h). Chase samples with trypsin for
BT62 are in lanes 7, 11, and 15.
Figure 1:
Growth of SecE depletion
strains. Strain CM124 was grown overnight in arabinose and then
passaged into medium containing either glucose () or arabinose
(
). BT62 has the same growth curve as CM124 (data not shown).
Under depletion conditions, cells grown in glucose can still recover
from the effects of the SecE depletion if switched to medium with
arabinose. There is a high plating efficiency (>50%) for cells moved
from minimal broth with glucose to LB agar with arabinose up to
5-7 h after the incubation in glucose has started. We never have
recovered any revertants of CM124 or BT62 that can grow continuously on
medium with glucose and without arabinose.
CM124 cultures were assayed for the extent of their secretion defect during the course of SecE depletion. At several time points after dilution into medium with glucose or arabinose, this secretion defect was visualized by pulse labeling cells and examining the ratio of precursor to mature form of several secreted proteins using immunoprecipitation and SDS-PAGE (Fig. 2). Cells expressing SecE (grown in arabinose) exhibit no accumulation of the precursor forms of the periplasmic proteins DegP and MBP or the outer membrane protein OmpA. In contrast, cells depleted of SecE exhibit a marked defect in secretion of these proteins after 3.5 h (a time at which the two cultures are doubling at the same rate), which becomes nearly complete at later time points. Precursor forms of exported proteins such as DegP, which had previously been difficult to observe under restrictive conditions for the conditional Sec mutants, were readily observed in the SecE-depleted cells. In addition, OmpA translocation during the course of a 5-min chase was substantially less efficient than that observed using Sec conditional mutants(13) .
Figure 2: Depletion of SecE results in a severe secretion defect. Strain CM124 was grown as described in Fig. 1. Pulse-chase labeling was performed at 3.5, 5.5, and 7.5 h. The translocation of MBP, DegP, and OmpA was assayed by visualization of immunoprecipitated precursor (p) and mature (m) forms of these proteins at the pulse (0 time point) and 1-, 2-, and 5-min chase time points. The precursor form of DegP appears as a diffuse doublet rather than a discreet band on these gels.
To
assay for MalF insertion using the proteolysis assay, spheroplasts were
derived from a SecE depletion strain BT62 (malE The amount of radiolabeled MalF detected in
the different samples does not reflect significant variation in the
recovery or the integrity of the spheroplasts in each case. A fraction
of each of the solublized cell extracts from the samples shown in Fig. 3A was examined by Western blot analysis (shown in Fig. 3B) with antiserum specific for MalK, a peripheral
cytoplasmic membrane protein that associates with the cytoplasmic face
of the membrane. A significant sample to sample variation in the
recovery of the spheroplasts after the experiment would be detected as
a loss of MalK signal on the Western blot in one sample in a set
compared with its partners. If any of the spheroplasted samples had
lysed during the experiment, the trypsin would have degraded the MalK
protein inside the cell. This would be evidenced by a decrease in the
intensity of the MalK signal in the samples with trypsin compared with
controls without trypsin. The amount of MalK detected in the different
samples of each set of spheroplasted samples is similar. In this
experiment, less MalK is expressed in the SecE-depleted BT62 cells
grown in glucose for 3.5 h than in the Sec Some polypeptides unrelated to MalF also are immunoprecipitated by
the MalF antiserum. These are shown in control samples from strain
KM1089 ( The secretion of
MBP appears to be more sensitive than the membrane insertion of MalF to
the effects of SecE depletion. Fig. 4shows that when the
background levels of precursor MBP or protease-resistant MalF under
Sec
Figure 4:
The effect of Sec inhibition on MBP and
MalF localization. The abundance of precursor MBP and
protease-resistant MalF is shown for after NaN
Two additional
observations come from comparing the pulse-labeled samples with those
that had been pulse-labeled and then chased. First, at time points when
there is a substantial secretion defect (as measured by the inhibition
of MBP export, Table 1), there is a dramatic increase in the
amount of degradation of MalF by endogenous proteases during the course
of the chase (comparing the pulse without trypsin with the chase
without trypsin samples at 3.5 h in glucose; Table 1, MalF
stability). We discuss this observation further below. Second, the
portion of the MalF that is protease-resistant decreases by the end of
the chase during Sec
To confirm the results of
McGovern and Beckwith(1) , we tested the protease sensitivity
of pulse-labeled MalF in the Mal
The ambiguities
created by using Mal As was the case in the SecE depletion experiments,
this uninserted population of MalF in NaN To show that the species of MalF that is sensitive to
endogenous proteolysis in NaN These results indicate
that the proper insertion of MalF requires the activity of SecA in
addition to SecE. As with the SecE-depleted cells, the inhibition of
MalF membrane insertion and MBP export caused by NaN The involvement of the Sec proteins in the export of signal
sequence-containing proteins such as OmpA and MBP is well documented.
However, a general mechanism for the insertion of proteins into the
cytoplasmic membrane, including translocation of their hydrophilic
domains across this membrane, has not been established. This issue has
been addressed in only a few cases, and the examined proteins fell into
two classes: 1) Sec-independent, e.g., M13 procoat, ProW, and
MalF, and 2) Sec-dependent, e.g., Lep, SecY, and
Tsr(1, 7, 8, 9, 15) . In
contrast to earlier work, we show here that MalF insertion into the
cytoplasmic membrane requires both SecE and SecA. Systems to study
the Sec dependence of cytoplasmic membrane protein insertion have been
plagued by two major problems. First, a general assay (such as signal
sequence cleavage for fully secreted proteins) for cytoplasmic membrane
protein insertion does not exist. To compensate for this, several
studies have used an indirect assay for Sec dependence that relies on a
comparison of the activity or localization of an enzymatic/polypeptide
tag in fusion protein constructs in Sec The second major pitfall encountered in previous studies was
the use of relatively weak conditional sec mutations. As has
been shown for secEcs mutations, many conditional sec mutations likely cause a decrease in the amount of functional
secretion apparatus in the cell(5, 13) . It seemed
possible to us that if cytoplasmic membrane proteins in general had a
greater affinity for the secretion apparatus than fully secreted
proteins, then their membrane insertion and export of hydrophilic
domains would be relatively less affected by the limited amount of
secretion apparatus than that of fully secreted proteins. In fact,
various members within the class of fully secreted proteins in E.
coli, such as DegP, are only slightly affected by partial
secretion defects in the cell; others, such as MBP, are extremely
sensitive to even slight Sec defects. To address this issue, we sought
to utilize a stronger conditional allele of the secE gene. One
such conditional SecE mutant, in which the secE gene is
expressed from the araBAD promoter, has been described in
previous studies(6) . Because SecE is an essential component of
the secretion apparatus, strains containing the p The secretion defect in cells that have been
depleted for the SecE protein is much stronger than that observed in
other conditional sec mutants. The magnitude of this defect is
evidenced by two findings. First, CM124 cells depleted of SecE
accumulate large amounts of precursor species of proteins such as DegP,
which have been difficult to visualize previously (Fig. 2).
Second, precursors of proteins, such as OmpA, that accumulate under
SecE depletion conditions are very slowly converted to the mature
species during the course of a chase. This is in contrast to the fate
of precursor OmpA accumulated in many other sec conditional
mutants where a large proportion of this precursor species eventually
becomes exported(13) . Conversely, there is no detectable
secretion defect in the SecE depletion strains when grown under
permissive conditions, in contrast to other secE conditional
alleles (Fig. 2; (13) ). The ability to both elicit a
strong secretion defect in SecE-depleted cells and to assay MalF
insertion in a sensitive manner led us to readdress whether MalF has a
Sec requirement for its insertion into the cytoplasmic membrane. In
the SecE depletion strain BT62 (malF The dependence of MalF on SecE for its membrane insertion
prompted us to re-examine the involvement of the SecA protein in this
process. Like SecE-depleted cells, NaN Previously, McGovern and Beckwith (1) had examined the
protease sensitivity of pulse-labeled MalF in cultures of a
Mal In both SecE-depleted and SecA-inhibited
(NaN The amount of
protease-resistant MalF caused by NaN This proposal is supported by work from
two groups characterizing the protein export (SEC) pathway in Saccharomyces cerevisiae. Green et al.(19) and Stirling et al.(20) found that
the insertion of proteins into the endoplasmic reticulum membrane is
dependent on several proteins, including SEC61 (a homolog of the E.
coli SecY protein). However, not all sec We have determined that MalF depends on the Sec apparatus for proper
membrane insertion using a direct protease assay coupled with two
strategies for eliciting potent and specific secretion defects. In
contrast, McGovern and Beckwith (1) concluded that MalF does
not utilize the Sec apparatus for membrane localization by examining
MalF and MalF fusion proteins in either SecDFcs mutants or
NaN Our study highlights the
pitfalls and difficulties in determining the Sec involvement in
integral membrane protein assembly. Our results suggest that great
caution should be used in the interpretation of localization data from
fusion protein studies, given that the MalF-PhoA fusions did not show a
SecA dependence clearly evident from our analysis of the intact protein
and from the analysis of some kinds of fusion proteins. The exclusive
use of NaN Further studies
are needed to determine whether membrane proteins without cleavable
signal sequences generally rely on the Sec pathway for membrane
assembly. Based on studies with an extensive collection of Lep fusion
constructs, von Heijne and co-workers have proposed that the
translocation of short (
F
G
K
). We chose time points early in the SecE
depletion process to maximize the likelihood that we were testing the
direct effects of SecE depletion on MalF membrane insertion. In
addition, the strength of the secretion defect for MBP was monitored in
parallel as described for Fig. 2, so that a comparison of
protein secretion versus cytoplasmic membrane protein
insertion could be made. In cells that are either Sec
or depleted of SecE for 2 h, the MalF protein was inserted into
the cytoplasmic membrane in a trypsin-sensitive form, with its
periplasmic domains accessible to protease (Table 1, +Ara
and +Glu, 2 h; +Ara samples shown in Fig. 3A, lanes 3-6). In contrast, at later time points of SecE
depletion, a substantial proportion of MalF became insensitive to
trypsin proteolysis (Table 1, +Glu, 2.75 and 3.5 h;
+Glu at 3.5 h shown in Fig. 3A, lanes
7-10). This MalF protein became trypsin-sensitive if the
spheroplasts in these experiments were lysed prior to the addition of
the protease (data not shown), indicating that this form of MalF was
not intrinsically protease-resistant. We speculate that in
secretion-compromised cells, MalF that is insensitive to externally
added protease is localized to one of two compartments. Because of the
hydrophobic nature of the transmembrane domains of MalF, uninserted
protein might aggregate in the cytoplasm. Alternatively, insertion of
some or all of the transmembrane domains might occur, resulting in MalF
that is apposed to or partially inserted into the cytoplasmic membrane.
This latter species would have some or all of its periplasmic domains
(that are normally sensitive to external protease) in the cytoplasm. To
exclude the possibility that SecE-depleted cells were resistant to
spheroplast formation, which would also result in protease-resistant
MalF, steady state populations of MalF were shown to be sensitive to
externally added protease by Western blot analysis (data not shown).
This population of MalF molecules presumably would have been properly
incorporated into the membrane during the Sec
conditions of the depletion time course and persisted to late
times in the experiment.
cells grown
in arabinose (Fig. 3B, lanes 5-7 versus
12-15). In this experiment, we also observed a 30-40%
decrease in the MalF and MBP expression under the same conditions (Fig. 3A, lanes 7 and 9 versus lanes 3 and 5 and data not shown). We found this overall decrease
in the expression of the mal genes under SecE-depleted
conditions in approximately half of the repetitions of this experiment.
malF; Fig. 3A, lanes 1, 2, 15, and 16), which does not express MalF.
The most notable of these are denoted as bands a, b, c, and d. The polypeptides c and d are only detected after proteolysis of SecE-depleted or
SecA-inhibited (see below) cells and are absent from samples from
Sec
cells. The identity of these polypeptides is
unknown. However, they do not interfere with the quantitation of the
protease sensitivity of MalF and do not represent novel MalF tryptic
peptides produced under Sec-inhibited conditions.
conditions (BT62 with arabinose) are subtracted,
the effect of a Sec defect on MBP export is detectable sooner and is
greater than the effect on MalF membrane insertion.
treatment or
at different time points in the SecE depletion time course for BT62.
The background level of protease-resistant MalF in cultures grown in
arabinose was subtracted from the data shown in Table 1.
conditions (Table 1,
+Glu, 2.75 and 3.5 h). The diminution of the secretion defect over
the course of the chase is also observed in the MBP analysis and
probably reflects residual Sec activity in the depleted cells.
Membrane Incorporation of MalF in Sodium Azide-treated
Cells
Previous work had suggested that MalF is not dependent on
the SecA component of the secretion apparatus for its insertion into
the cytoplasmic membrane(1) . In these experiments, cells were
treated with NaN to inhibit SecA function(14) , and
then MalF insertion was assayed by a protease sensitivity assay similar
to the one described here. Two important facts led us to re-examine the
dependence of MalF insertion on SecA. First, as described in the
previous section, MalF insertion was dependent on expression of SecE,
one of the core components of the secretion apparatus. Because the
basic mechanism of secretion is thought to involve the actions of SecE,
SecY, and SecA, it seemed likely that SecA might also be involved in
MalF insertion. Second, the experiments of McGovern and Beckwith (1) were performed in a Mal
strain background.
Normally, MalF expressed in a Mal
strain acquires a
protease-resistant form that reflects its inclusion into a stable
complex with MalG and MalK; absence of either of these components of
the complex results in protease-sensitive MalF(10) . It is
possible that in the Mal
strains, MalF could acquire
protease resistance by two different mechanisms, one in which MalF
becomes complexed with MalG and MalK and one in which MalF is not
inserted into the membrane due to a secretion defect. To address this
latter possibility, both Mal
and Mal
strains were used to assess NaN
sensitivity of MalF
insertion into the cytoplasmic membrane.
strain BT8 with and
without treatment with NaN
. Without NaN
treatment, MalF initially inserts into the m
embrane in a
trypsin-sensitive form and is rapidly converted into a
trypsin-resistant conformation as a consequence of complex assembly
with MalG and MalK (Table 2). The initial trypsin-sensitive form
is detectable as a portion (roughly half) of the population of labeled
MalF molecules after a short pulse time point in this experiment; the
MalGK-complexed, trypsin-resistant MalF form is already abundant at the
pulse time point and becomes the predominant species after a chase (Table 2). Consistent with previous results, cells treated with
NaN
show no difference in the amount of trypsin-resistant
MalF at the pulse time point compared with nontreated cells. In
contrast, our added quantitation of the amount of trypsin-resistant
MalF after a 10-min chase in the NaN
-treated versus untreated samples showed a significant difference (35 versus 65%). The inability to distinguish betwee
n the two possible
populations (described above) of trypsin-resistant MalF in these
Mal
cells makes interpretation of the data difficult.
However, the lower level of protease-resistant MalF at the end of the
chase in NaN
-treated Mal
cells suggests
that the ability of MalF to assemble into the MalFGK complex in the
membrane was compromised in the treated cultures.
cells were alleviated by an
analysis of the protease sensitivity of MalF in the similar malG
strains BT62 and BT10. (For comparison
with the SecE depletion data, the data from NaN
-treated
BT62 cells are shown in Fig. 3and Fig. 4and in Table 1. The quantitation of the BT10 data is given in Table 2. NaN
-treated control samples of the
malF strain KM1089 are shown in Fig. 3.) In the
cultures not treated with NaN
, the majority of MalF
synthesized in a pulse is inserted into the cytoplasmic membrane in a
trypsin-sensitive form, consistent with previous results(10) .
This population of MalF remains trypsin-sensitive after chase, because
it is unable to form a native complex. In NaN
-treated
cultures, 30-40% of the MalF synthesized during a pulse-labeling
is resistant to exogenous trypsin. Because none of this resistant
species is attributable to MalFGK complex formation, it represents
noninserted MalF.
-treated cells was
susceptible to endogenous proteases, as evidenced by its gradual
disappearance during the course of a 10-min chase (Table 2). The
stability of MalF over the course of a 10-min chase in
NaN
-treated cultures ranges from 48-68%, compared
with a slight increase in the amount of MalF detected in the chase
compared with the pulse time points in untreated Sec
cells. The decrease in MalF stability in Sec-inhibited cells is
observed in both Mal
and the malG
strains.
-treated cells was actually
the result of a block in SecA activity, we characterized the stability
of the protein in the Mal
strains BT7 and BT63. BT7
produces a wild type SecA protein. BT63 produces a SecA mutant that is
partially resistant to NaN
(from the azi-4 allele; (14) ). The trypsin sensitivity of MalF in pulse-labeled
cultures of BT7 with and without NaN
treatment is analogous
to that observed in BT8 under similar conditions (Table 2).
However, the sensitivity of MalF to trypsin and endogenous proteases in
BT63 is relatively unchanged by the presence or the absence of
NaN
treatment at both the pulse and the chase time points.
The NaN
treatment of BT63 apparently did not impair either
the membrane insertion of MalF or its subsequent assembly into the
MalFGK complex. This result strongly suggests that the NaN
treatment of azide-sensitive cells affects the proper membrane
insertion of MalF and the stability of MalF to proteolysis directly via
the inhibition of SecA protein and not indirectly by a general
disruption of the cell's physiology.
is
diminished at the end of a 10-min chase after pulse-labeling. The
inhibition of membrane insertion for MalF is not as strong with
NaN
as with SecE depletion at the 3.5 h time point ( Fig. 4and Table 1). However, the inhibition of MBP export
is as strong with NaN
as with SecE depletion in BT62 at 3.5
h.
or
Sec
conditions(8, 9) . Although the
results from these experiments are suggestive, the notion that the
marker tag of the fusion protein does not affect the normal process of
membrane insertion for the target protein is largely untested. A few
specific proteins can be directly tested for Sec involvement in
membrane insertion. For example, the studies on the membrane insertion
of Lep assay its Sec dependence for insertion by the protease
sensitivity of a large periplasmic domain in Sec-inhibited
spheroplasts(7) . The inhibition of export of this domain
results in the accumulation of protease-resistant Lep. Unfortunately,
relatively few integral cytoplasmic membrane proteins can be analyzed
by this method because of their general resistance to exogenous
proteases in extracytoplasmic domains (e.g., Tsr; (16) ). MalF rapidly acquires resistance to exogenous proteases
upon its export to the cytoplasmic membrane when assembled into the
oligomeric MalFGK complex. However, in spheroplasted MalG
cells, properly localized MalF is sensitive to added
proteases(10) . This finding allows for a sensitive MalF
insertion assay in which one can attribute protease-resistant MalF in malG
cells unambiguously to noninserted
MalF.
secE construct are absolutely dependent on arabinose for prolonged
growth (Fig. 1).
malG
) grown
in arabinose, MalF is inserted into the membrane and is sensitive to
trypsin in spheroplasted cells ( Fig. 3and Table 1). This
trypsin sensitivity is indistinguishable from that observed in wild
type secE
malF
malG
cells (Table 1, Table 2; also see 10). In cells depleted of SecE
for 3.5 h, there is a dramatic increase in the amount of pulse-labeled
MalF protein that is resistant to proteolysis (Fig. 4). These
data strongly suggest that MalF requires the SecE component of the
secretion apparatus for its proper insertion into the cytoplasmic
membrane.
-treated BT62 cells
also contain an elevated level of trypsin-resistant MalF immediately
after the synthesis of the protein ( Fig. 3and Table 1).
This elevated level of trypsin-resistant MalF is also seen in the secE
malF
malG
strain BT10 when treated with
NaNa
(Table 2). These results are consistent with an
additional dependence on SecA for the membrane insertion of MalF.
strain with and without treatment with
NaN
(1) . In both cases, they observed a population
of protease-sensitive MalF (
50%) giving rise to similar peptide
patterns after SDS-PAGE. To directly compare our results with theirs,
we examined the protease sensitivity of MalF in the Mal
strain BT8 (Table 2). We found that a similar amount of
trypsin-resistant MalF is present at the pulse time point, with or
without NaN
treatment, consistent with the observations of
McGovern and Beckwith(1) . We further characterized the amount
of MalF and its trypsin sensitivity after a chase. The increase in the
level of trypsin-resistant MalF during a chase in
NaN
-untreated cells reflects the continued incorporation of
MalF into the MalFGK complex. In contrast, there is a decrease in the
amount of trypsin-resistant MalF at the end of a chase in the presence
of NaN
. Therefore, proteolysis experiments that only
examined the state of MalF at the pulse time point in Mal
cells led to incorrect conclusions regarding the Sec dependence
of MalF.
-treated) cultures, an unstable population of MalF is
revealed in the unproteolyzed controls (see Table 1and Table 2, MalF stability). This turnover of newly synthesized
protein during the course of the chase is a dramatic increase in the
sensitivity of MalF to endogenous proteases. We suggest that
when MalF is not properly inserted into the membrane, it becomes a
substrate for cellular proteases, which recognize it as a misfolded
protein. In BT63, containing NaN
-resistant SecA, NaN
treatment did not result in a l
oss in MalF stability to
endogenous proteases (Table 2).
inhibition of SecA is
smaller than that caused by SecE depletion of the cell ( Fig. 4and Table 1). This difference in the inhibition of
the membrane insertion of MalF caused by the different treatments is in
contrast to a similar inhibition of MBP export due to NaN
treatment compared with SecE depletion at 3.5 h +Glu (Fig. 4). MalF also seems be affe
cted at later times than MBP in
the SecE depletion time course. The greater defect in the membrane
insertion of MalF caused by SecE depletion than by NaN
inhibition of SecA is consistent with two previous observations.
First, Werner et al.(17) have observed in vitro that the cytoplasmic membrane mannitol carrier protein MtlA was
dependent upon the SecY protein for proper membrane insertion. However,
MtlA inserted into membranes normally in the presence of reduced levels
of SecA. Second, Rusch et al.(18) have characterized
the export of alkaline phosphatase precursors as a function of the
relative hydrophobicity of their signal sequences. They found that
signal sequences with very high hydrophobicity mediated precursor
export that was not detectably inhibited by treatment of cells with
NaN
. Proteins localized by the Sec apparatus with very hydrophobic signal sequences or transmembrane domains (like MalF
or MtlA) may have a higher affinity for the Sec machinery than more
typical signal sequence-containing precursor proteins. Therefore,
detecting a Sec dependence for this class of proteins may require a
severe Sec defect stronger than most conditional Sec mutants at
nonpermissive conditions.
alleles can result in inhibition of membrane protein insertion.
Apparently strongly defective mutants or double mutants are required in
many instances to detect a SEC involvement in membrane protein
insertion, whereas weaker mutants will inhibit the export of several
soluble proteins with cleavable signal sequences across the membrane.
-treated cells. Our work does not address the dependence
of MalF on SecDF for translocation of periplasmic domains. We did assay
the export of periplasmic domains of MalF-PhoA fusion proteins in
NaN
-treated cells (data not shown). In results similar to
those of McGovern and Beckwith(1) , we also failed to detect
any inhibition caused by NaN
in the membrane assembly of
the MalF fusion proteins. However, recent experiments of
Sääf et al.(21) and Jander et al.(22) did
demonstrate a Sec dependence for export of some periplasmic domains in
other MalF fusion constructs. In the experiments of
Sääf et
al.(21) , NaN
treatment of cells expressing
MalF-Lep fusions caused a weak defect in export of the large
periplasmic domain of MalF, compared with the export of the precursor
form of OmpA. The work of Jander et al.(22) demonstrated a role for both SecA and SecE in the export
of biotinylated tags fused to different regions of Mal, but the degree
of Sec involvement was not quantified.
-inhibition of SecA as a secretion defect in this
kind of experiment may be inadequate. We advise the additional use of
strong Sec mutants (such as a SecE depletion strain) for the analysis
of Sec dependence for protein localization studies.
60 residues) extracytoplasmic domains is
Sec-independent, whereas that of longer domains is generally Sec-dependent (reviewed in (23) ). The membrane insertion
of MalF is now partially consistent with this model. However, because
we quantified the amount of intact MalF after proteolysis, our
results suggest that MalF may require the Sec machinery for
translocation of each of its three periplasmic domains (with 180, 30,
and 30 residues) that are susceptible to proteolytic cleavage, not just
the largest. It may be that many, if not most, other membrane proteins
will exploit the Sec machinery during the normal in vivo insertion and assembly process, regardless of the size of the
hydrophilic domains to be translocated.