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J. Biol. Chem., Vol. 275, Issue 31, 24191-24198, August 4, 2000
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From the
Received for publication, April 5, 2000
During localization to the periplasmic space or
to the outer membrane of Escherichia coli some proteins are
dependent on binding to the cytosolic chaperone SecB, which in turn is
targeted to the membrane by specific interaction with SecA, a
peripheral component of the translocase. Five variant forms of SecB,
previously demonstrated to be defective in mediating export in
vivo (Gannon, P. M., and Kumamoto, C. A. (1993)
J. Biol. Chem. 268, 1590-1595; Kimsey, H. K.,
Dagarag, M. D., and Kumamoto, C. A. (1995) J. Biol. Chem. 270, 22831-22835) were investigated with respect to
their ability to bind SecA both in solution and at the membrane
translocase. We present evidence that at least two regions of SecA are
involved in the formation of active complexes with SecB. The variant
forms of SecB were all capable of interacting with SecA in solution to
form complexes with stability similar to that of complexes between SecA
and wild-type SecB. However, the variant forms were defective in
interaction with a separate region of SecA, which was shown to trigger
a change that was correlated to activation of the complex. The region
of SecA involved in activation of the complexes was defined as the
extreme carboxyl-terminal 21 aminoacyl residues.
Export of protein from the cytosol of Escherichia coli
to the periplasm and to the outer membrane is mediated by a
membrane-bound translocase, which comprises a peripheral ATPase, SecA,
and two integral membrane heterotrimeric protein complexes: SecYEG and a complex of SecD, SecF, and YajC. Some of the precursor proteins translocated by this apparatus are targeted to the membrane by interaction with the cytosolic chaperone SecB, which has affinity for
SecA (for review see Driessen et al. (1)). Analyses of mutant strains, defective in export (2, 3), have led to the
identification of variant forms of SecB that are altered in oligomeric
structure and in interaction with precursor proteins (4) or in the
interaction with SecA (5). Here we have analyzed variants that are
defective in binding SecA. We have demonstrated that wild-type SecB
forms two types of complexes with wild-type SecA in solution and binds
the membrane translocase in a functional manner. Each of the SecB
variants was capable of making only one of these complexes, and that
complex did not bind to membrane. A truncated form of SecA, missing the
21 carboxyl-terminal residues and identified previously as the sole
binding site for SecB (6), was shown to retain the ability to bind both
wild-type and variant forms of SecB, but all the complexes formed were
nonfunctional. Thus there are at least two separate regions of SecA
that are involved in the formation of active complexes with SecB. The
interactions at sites as yet unidentified but common to the wild-type
and variant forms of SecB provide sufficient energy to stabilize the
complexes. The interaction between SecB and the extreme
carboxyl-terminal region of SecA, which is defective for the variants
of SecB, plays a regulatory role in conversion of complexes to an
active state.
Bacterial Strains and Plasmids--
E. coli strain
HB1215 is an OmpT Protein Purification--
Mature galactose-binding protein (11),
precursor galactose-binding protein (12), SecB, and the five variants
of SecB (4, 13) were purified as described. SecA and SecAN880 were
purified from strains RR1/pMAN400 (10) and HB1732,
respectively. Cells were grown in M9 minimal medium (14) supplemented
with 0.4% glucose, 4 µg/ml thiamine, 1% casamino acids, and 100 µg/ml ampicillin at 35 °C to an optical density at 560 nm of 0.8;
0.2 mM isopropyl-1-thio-
Concentrations of purified proteins were determined
spectrophotometrically at 280 nm using coefficients of extinction as
follows: SecB and SecB variants, 47,600 M Purification of Radiolabeled SecB--
14C-SecB was
purified from strain BL21( Size-exclusion Chromatography--
Chromatography was carried
out using a TSK G3000SW (TosoHaas) size-exclusion chromatography column
(7.5-mm inner diameter × 60 cm) equilibrated in 10 mM
HEPES, 1 mM EGTA, 300 mM KOAc, 5 mM
MgOAc, 2 mM DTT, pH 7.5. Samples of 200 µl were prepared in the same buffer, separation was carried out at 8 °C at either 0.9 or 1 ml/min, and absorbance was monitored at 280 nm. The slight variation of the elution positions of the proteins that is seen upon
comparison of the Figures is the result of using two different TSK
G3000SW columns during the course of this work. The profiles shown in
Figs. 1, 3B, 3C, 4, and 9B were
obtained using one column, and those in Figs. 2, 3A, and
9A were obtained using the other column.
Preparation of Inverted Cytoplasmic Membrane
Vesicles--
Bacterial cultures (HB1215 or HB1590) were grown in
Luria broth (14) in the presence of appropriate antibiotics to an
absorbance of 0.7 at 560 nm, induced with 1.5 mM IPTG, and
harvested 2 h later. Vesicles were prepared as described (16),
suspended in 10 mM HEPES, pH 7.6, 150 mM KOAc,
1 mM DTT, 250 mM sucrose to give a protein
concentration of 6-9 mg/ml as determined by the bicinchoninic acid
assay (Pierce), and stored at 14C-SecB Binding Assays--
Binding assays were
carried out in 125 µl of 10 mM HEPES, pH 7.6, 300 mM KOAc, 2 mM DTT, 1 mM EGTA, 50 mM guanidine hydrochloride (GdnHCl), 20 mM
sucrose, HB1590 vesicles containing 75 µg of protein, and where
indicated, 5 mM MgOAc, and 2 mM ATP. The
concentration of metabolically labeled 14C-SecB (wild-type,
L75Q, or E77K) was 1 µM. When present, the concentration
of SecA and precursor galactose-binding protein was 1 µM
each unless indicated otherwise. Mixtures were incubated on ice for 15 min. Vesicles were then pelleted by centrifugation (Beckman L8-70
ultracentrifuge) in a 42.2 Ti rotor at 14,000 rpm for 21 min
( In Vitro Processing of Precursor Galactose-binding
Protein--
Processing of precursor galactose-binding protein by
vesicles prepared from HB1215 was performed in a 30-µl reaction
mixture containing 10 mM HEPES, pH 7.0, 300 mM
KOAc, 2 mM DTT, 0.7 mM EGTA, 6.25 mM magnesium acetate (MgOAc), 2.5 mM ATP, and 2 µM SecB unless indicated otherwise. Unfolded precursor
galactose-binding protein was diluted 15-fold from a 30 µM stock (the final GdnHCl concentration varying from 50 to 75 mM) into the mixture. SecA (2 µM) was
added, and the translocation reaction was initiated by the addition of
vesicles containing 19-38 µg of proteins. Samples were incubated at
37 °C for the indicated times and transferred to ice, and an equal
volume of 2-fold-concentrated electrophoresis sample buffer was added.
After boiling for 5 min, the samples were analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblotting. The amount
of mature galactose-binding protein in each sample was determined by
quantifying the immunoblot using a densitometer (Molecular Dynamics,
Inc.) and relating the intensity of the band representing mature
galactose-binding protein to a standard curve of pure authentic mature
galactose-binding protein on the same immunoblot.
Gel Electrophoresis and Immunoblotting--
SDS-polyacrylamide
(10%) gel electrophoresis was performed as described (17).
Immunoblotting was performed as described (4) using an antiserum to
galactose-binding protein.
Mutational studies of SecB have led to the identification of
substitutions that result in defects in protein export (2, 3). The
substitution mutations cluster in two regions, one near the amino
terminus at residues Asp-20 and Glu-24, and a second region between
aminoacyl residues 73 and 81. The altered forms of SecB have been
classified into two groups based on the functional defect exhibited
(3). One group, comprising those with substitutions at even-numbered
residues between 73 and 81, was originally characterized as having a
reduced ability to form complexes with precursor proteins (3) and
recently has been shown to be altered in the oligomeric state of SecB
(4). The second group, comprising substitutions at Asp-20, Glu-24,
Leu-75, and Glu-77, was shown to interact normally with precursor
protein ligands as assessed by coimmunoprecipitation of SecB with
precursor maltose-binding protein (Asp-20 and Glu-24 (3)) as well as by
direct binding assays (Leu-75 and Glu-77 (4)). Two members of that
group, those having substitutions at Leu-75 and Glu-77, were further
investigated and shown to be defective in interaction with
membrane-associated SecA (5). To determine the molecular basis of the
inability to sustain protein export, which is reflected in the
defective membrane interaction, we have extended the analysis of the
two previously characterized variants of SecB to include examination of
their interactions with SecA free in solution. We have also
investigated three additional variant forms of SecB. It is important to
examine the interactions in solution as well as at the membrane,
because under physiological conditions in vivo soluble
complexes are expected to form. There is an excess of SecA relative to
its binding sites at the membrane translocase, and the concentrations
of both the cytoplasmic SecA and SecB (estimated to be approximately 4 and 20 µM,
respectively2) are above the
dissociation constant that has been determined for the SecA·SecB
complex in solution (Kd approximately 1.6 µM (18)). Furthermore, soluble ternary complexes of SecA, SecB, and precursor proteins have been isolated from cellular lysates
(19). It is also of interest in this context to note that the
membrane-associated SecA has been shown to exist in vivo in
two different conformations, one of which is exchangeable with soluble
SecA (20, 21). Therefore, although it is not clear whether or not
formation of complexes between SecA and SecB in the cytoplasm is a
necessary step in delivery of SecB and its precursor protein ligand to
the translocase, characterization of the soluble complexes should help
to elucidate the nature of the interaction at the membrane.
Analyses of Ligand Binding by SecB Variants--
The ability to
bind precursor galactose-binding protein, a natural ligand of SecB, has
been assessed previously (4) using purified proteins for two of the
variant forms of SecB under study here: SecBL75Q (the leucine at
position 75 is replaced by glutamine) and SecBE77K (the glutamate at
position 77 is replaced by lysine). The binding was shown to be
indistinguishable from that between precursor galactose-binding protein
and wild-type SecB by two methods: analysis of complex formation by
size-exclusion chromatography and by titration calorimetry. Here we
demonstrate that the three previously uncharacterized variants that
have aminoacyl substitutions in a different region, SecBD20A, SecBE24A,
and SecBE24K, also show normal interaction with precursor
galactose-binding protein. SecB only binds proteins as ligands if they
are in a non-native state (22). Therefore, to assess interaction
precursor galactose-binding protein was unfolded in guanidinium
chloride and refolding was initiated by dilution of the denaturant in
the presence of SecB. Equimolar mixtures of SecB and precursor
galactose-binding protein were analyzed by size-exclusion
chromatography. In all cases whether the SecB were wild-type (Fig.
1, dotted line) or one of the
variants, SecBE24A (Fig. 1, solid line), SecBE24K (Fig. 1,
short-dashed line), or SecBD20A (data not shown) the
galactose-binding protein coeluted with SecB, ahead of the positions of
free SecB (15.8 ml) and free precursor galactose-binding protein (17.2 ml). We conclude that these variants, like SecBL75Q and SecBE77K, are unaltered in their oligomeric state (they elute at 15.8 ml as does
wild-type) and with respect to binding of ligand. We next investigated
all five variants for their ability to bind SecA.
Analyses of Complexes between SecA and SecB Formed in
Solution--
Direct demonstration of the formation of complexes
between SecB and SecA free in solution was achieved using
size-exclusion chromatography. When applied separately to a TSK G3000SW
column, SecB and SecA elute at different positions (Fig.
2, 15.2 ml for SecA (dotted
line), and 15.8 ml for SecB (short-dashed line)). When
equimolar mixtures of SecB tetramer and SecA dimer at 1 µM (Fig. 2, dotted-dashed line), 4 µM (Fig. 2, solid line), or 6 µM
(Fig. 1, long-dashed line) were applied to the column, the SecB and SecA formed complexes as evidenced by the fact that the material absorbing at 280 nm eluted earlier than did either protein alone. Examination by SDS-polyacrylamide gel electrophoresis of the
protein present in each fraction shows that the material eluting between 13 and 15 ml contains both SecA and SecB. The complexes elute
earlier, i.e. at higher apparent molecular weights, as the concentrations of proteins applied are increased. This behavior is
characteristic of species in equilibrium and is consistent with the
previously published determination of the dissociation constant of the
SecA·SecB complex as being in the micromolar range (18). Thus, when a
mixture of the proteins each at 1 µM was applied, the
dilution that occurred during chromatography resulted in the
concentrations being well below the Kd level, and
therefore, the distribution of the material reflected very little
complex formation (Fig. 2, dotted-dashed line). Application of samples at concentrations above the Kd level
resulted in a shift in the equilibrium toward the complexed state.
Because the position of elution of the material reflects the weight
average of the position of elution of the different species, which are constantly re-equilibrating during chromatography, the peak was observed to move forward as the concentration at which the samples were
applied increased. Careful examination of the absorbance profiles of
the complexes indicates the presence of at least two species of complex
as revealed by the distinct shoulder discernable between the earliest
eluting peak and the latest (representing uncomplexed SecA and/or
SecB).
We examined the ability of variant forms of SecB to make complexes with
SecA. Two of the variants, SecBL75Q and SecBE77K have been reported to
be defective in binding to membrane vesicles through interaction with
SecA (5), but they had not been tested for binding free in solution.
The interaction of SecA with the other three variants, SecBD20A,
SecBE24A, and SecBE24K, has not been examined either in solution or at
the membrane translocase. All five species formed complexes with SecA
in solution as assessed by size-exclusion column chromatography. Fig.
3A shows that the complex
formed with SecBD20A differed from that formed between SecA and
wild-type SecB in that only one species of complex appeared to be
present and it eluted at 14.7 ml, a position that corresponds to the
shoulder in the elution pattern of the complexes formed between SecA
and wild-type SecB (Fig. 3A; compare the solid
(SecBD20A) and dotted lines (SecB wild-type)). Examination
of the interaction of SecA with SecBE24A, SecBE24K, SecBL75Q, and
SecBE77K (Fig. 3, B and C) showed that each
variant formed one species of complex eluting at the same position as
that seen for SecBD20A (Fig. 3A). We have designated the
complex that is formed by both the wild-type SecB and the variants as
complex 1 (eluting at 14.7 ml), and the species that is formed only
with wild-type SecB (eluting earlier, at around 13.5 ml when applied at
4 µM) as complex 2. We are justified in considering the
complexes designated complex 1 as closely related whether formed by
wild-type or variant SecB species, because two of the SecB variants,
SecBL75Q (Fig. 4) and SecBE77K (data not shown), were shown to compete with radiolabeled wild-type SecB for the
SecA in complex 1 but not in complex 2.
Addition of precursor galactose-binding protein to the mixtures of SecA
and SecB resulted in formation of ternary complexes, which contained
the precursor and eluted even earlier from the column. However, the
pattern observed in the absence of precursor was maintained: the
complexes containing wild-type SecB eluted earlier than did those
formed by the variants (data not shown).
Binding of SecB Variants to Membrane Vesicles via Interaction with
SecA--
The published studies demonstrating that SecBL75Q and
SecBE77K were defective in binding to inverted vesicles via association with SecA were carried out with SecA at 250 nM (5) (the
Kd for the interaction of SecB and membrane-bound
SecA is approximately 30 nM (6, 23)). Thus, because this
concentration is too low to allow formation of the soluble complexes
under study here, it was necessary to re-examine the binding to
inverted vesicles. In our assay both SecA and SecB were present in the
micromolar concentration range that would allow complexes to form in
solution so that we could determine whether these complexes would bind the translocase. Under these conditions, the binding of wild-type SecB
(1 µM) to inverted vesicles that contained the SecYEG
translocase was dependent on the addition of SecA (Fig.
5). Approximately 16% of radiolabeled
SecB (corresponding to 0.16 µM SecB) pelleted with
vesicles when SecA was added. In the absence of SecA only 3% of the
SecB was membrane-associated. Addition of precursor galactose-binding
protein to the system increased the binding of SecB to the membrane to
30% (Fig. 5), whereas addition of mature galactose-binding protein did
not affect the binding (data not shown). This is consistent with
previously published observations that the presence of a precursor
protein in the complex enhanced binding at the membrane translocase
relative to the binding of the SecB·SecA complex alone (5, 23).
Assessed under the same conditions (Fig. 5), the two variant SecB
species showed very little SecA-dependent binding in the
absence of precursor galactose-binding protein. Addition of
precursor galactose-binding protein stimulated binding of
SecBL75Q but not of SecBE77K. The lack of effect of the precursor on
the binding of SecBE77K was unexpected, because both SecBL75Q and
SecBE77K show normal binding of precursor galactose-binding protein
(4), and in the previous study by Fekkes et al. (5) the
ability of SecBE77K to compete with wild-type for membrane-bound SecA
was enhanced by proOmpA, a natural ligand of SecB. If the defect in
binding to membrane were one of affinity, then it would be overcome by
increasing the concentration of variant forms of SecB. Fig.
6 shows that this is not the case.
Although binding to membrane vesicles of complexes containing wild-type
SecB was saturated at 2.0 µM, there was no detectable
binding of either of the variants even at 10 µM SecB.
These results show that the defect in binding of the variant forms of
SecB to the membrane via SecA persists even at concentrations of the
proteins that should result in formation of soluble complexes.
Translocation and Processing of Precursor Galactose-binding
Protein--
In vivo SecB is required for efficient export
of its ligands, because it modulates a kinetic partitioning of the
precursor polypeptides between folding in the cytosol and entering the
export pathway (24). The binding to SecB is rapidly and readily
reversible and thus the ligand continuously samples the free state
(22). Because the variant forms of SecB bind their ligands normally and
thereby maintain them in an export competent state, it was possible
that the ligands could be passed to SecA directly without the necessity
for a complex containing SecA and SecB to bind to the membrane. To
eliminate this possibility, it was necessary to demonstrate that the
variant forms of SecB in the soluble complexes could not mediate
translocation, whereas the wild-type SecB could. The first step toward
this goal was a demonstration that a kinetic partitioning between
proteolytic maturation and folding does occur in our in
vitro protein translocation system and accurately mimics SecB-dependent export in vivo. It should be
noted that, just as in the binding assay used in this study, the
translocation system described here differs from published systems that
are used to translocate radiolabeled precursors present at very low
concentrations. Our assay contains the SecA, SecB, and precursor
galactose-binding protein at concentrations in the micromolar range so
that soluble complexes would be present. The amount of precursor
galactose-binding protein that is proteolytically processed to the
mature form is sufficiently high that it can be detected by staining
with Coomassie Blue and can be quantified by immunoblotting using an
antiserum raised to galactose-binding protein. We have previously shown that in the absence of Ca2+ the folding of precursor
galactose-binding protein is so slow that, with each cycle of
dissociation from SecB, the precursor galactose-binding protein rebinds
before it folds (12). However, if Ca2+ is present the rate
of folding is increased sufficiently so that the probability of
precursor galactose-binding protein refolding is higher than the
probability of its rebinding to SecB (12). Thus, in the presence of
Ca2+ the precursor partitions to the folded state, which is
not competent for translocation. Here we used Ca2+ to
modulate the rate of folding of precursor galactose-binding protein to
show that the processing to the mature form in the in vitro
assay is subject to a kinetic partitioning modulated by SecB. When the
folding of the precursor galactose-binding protein was slowed by
dilution from denaturant into the translocation buffer containing 0.7 mM EGTA, the same level of processing of the precursor to
the mature form was observed whether SecB, SecA, and inverted vesicles
were added immediately or after a 10-min incubation at 24 °C to
allow time for folding of the precursor (Fig.
7). Under these conditions, when folding
of the precursor is very slow, a low level of processing occurs even in
the absence of SecB. However, if the translocation buffer contained
Ca2+ in place of the EGTA to shift the kinetic partitioning
to favor folding, processing was greatly diminished even in the
presence of SecB. The processing was completely eliminated if the
translocation reaction was carried out without addition of SecB,
indicating that when the protein folds rapidly in the presence of
Ca2+ it is absolutely dependent on SecB for translocation.
These results are what is to be expected if the in vitro
translocation system accurately reflects the in vivo
functioning of SecB and involves a kinetic partitioning between folding
and translocation. Because neither SecBL75Q (data not shown) nor
SecBE77K (Fig. 8) supported processing of
precursor galactose-binding protein at any concentration tested, we
conclude that, although they can mediate a kinetic partitioning to
maintain the precursor in an unfolded state, the precursor cannot bind
SecA directly. Thus, neither SecBL75Q nor SecBE77K can support
translocation even at concentrations that result in formation of
complexes in solution.
Regulatory Interaction between SecB and the Carboxyl Terminus of
SecA--
All five SecB variants, SecBD20A, SecBE24A, SecBE24K,
SecBL75Q, and SecBE77K formed complex 1 with SecA and yet did not form complex 2 at all. Therefore, we considered the possibility that the
conversion from the inactive complex 1 to the active complex 2 is
triggered by an interaction between wild-type SecB and SecA that is
missing or defective when SecA binds the variant forms. A likely
candidate for a region of SecA that might act as the trigger was the
carboxyl-terminal 22 aminoacyl residues. Fekkes et al. (5)
had shown that this sequence from SecA fused to glutathione
S-transferase bound to wild-type SecB but did not bind
SecBL75Q or SecBE77K. In addition it has been shown previously (6) that
a form of SecA, which was truncated to remove the last 21 aminoacyl
residues (SecAN880), was incapable of mediating binding of SecB to
membrane vesicles. If the carboxyl-terminal region were indeed the
trigger that allowed conversion of complex 1 to an active species, then
we would predict that the truncated SecA would form complex 1 but not
complex 2 with SecB, whether the SecB were wild-type or a variant. Fig.
9 shows that this is the case. Truncated
SecA incubated with wild-type SecB or with SecBL75Q binds to yield
complex 1. We conclude that interaction between SecB and the carboxyl
terminus of SecA is crucial to trigger conversion of the inactive
complex 1 to complex 2. To further correlate the inactive state with
complex 1 we demonstrated that wild-type SecB in complex with SecAN880
does not bind to the membrane (data not shown) even at the micromolar
concentrations used in our assay as described for the variant forms of
SecB and wild-type SecA.
As discussed in Murén et al. (4), substitutions
in SecB at positions 76 and 78 disrupt the quaternary structure so that SecB, which is normally tetrameric, exists in solution as a dimer. The
variants studied here with substitutions at positions 20, 24, 75, and
77 have no detectable alteration in quaternary structure or in binding
to precursor polypeptides (4, and this report) but are affected in the
interaction of SecB with SecA. These variant forms of SecB interacted
with SecA in solution with an affinity similar to that of wild-type
SecB, but the species of complex formed, designated complex 1, was
defective in binding to inverted membrane vesicles and in mediating
processing of a natural ligand, precursor galactose-binding protein to
the matured form. The wild-type SecB, which formed complex 2, was
capable of binding to inverted membrane vesicles and mediating
processing of precursor. Because the truncated SecA formed the inactive
complex (complex 1) with all species of SecB tested, including the
wild-type SecB, we conclude that the carboxyl-terminal region of SecA
is not involved in interactions that provide the majority of energy to
stabilize that complex; rather, because the complex is inactive, the
interaction between SecB and the carboxyl terminus of SecA has a role
in triggering conversion of the inactive complex to an active state. It
was previously concluded (5, 6, 25) that the sole interaction between
SecA and SecB is confined to this carboxyl-terminal 21 aminoacyl
residues of SecA and that the variant forms of SecB are defective in
this interaction and therefore do not bind to SecA. These conclusions
were based on two experimental approaches. In one approach (5, 6), a
protein comprising glutathione S-transferase fused to the 22 aminoacyl residues from the carboxyl terminus of SecA was shown to bind
to wild-type SecB but not the variant forms. This result indicates only
that this aminoacyl sequence is a site of interaction but it need not
be the only binding site. Therefore, this is not in conflict with our
results, which implicate this region as a site of regulatory
interaction. Another study (5) assessed the binding of SecB to membrane vesicles via SecA but did not include investigations of interactions in
solution. Thus, that work is not inconsistent with our conclusions that
the variant species do form a complex with SecA in solution and that
the SecA in those complexes is in a state that is not capable of
interaction with the membrane.
Soluble complexes would be expected to exist in vivo,
because the binding sites at the membrane-associated translocase are saturated and the excess SecA and SecB are present in the cytoplasm at
concentrations well above the Kd level for the
interaction in solution (18). Complexes between the defective variants
of SecB and cytosolic SecA might trap SecA in a nonfunctional state. This could explain the dominant phenotype described for SecBL75Q (2)
and the fact that the export defect caused by SecBL75Q can be
suppressed by overproduction of SecA (26).
The apparent affinity of SecB for SecA bound to the membrane
translocase is higher (Kd, 30 nM (6,
23)) than is the affinity of SecB for SecA free in solution
(Kd, 1.6 µM (18, 23)). This suggests
either that SecB makes contacts with other membrane-associated
components in addition to SecA to provide increased binding energy or
that the membrane-associated SecA is in a different conformational
state than that free in solution. A change in conformation of SecA
might be elicited by binding to SecY in the translocase, and this form
could have high affinity for SecB. It is also possible that SecB itself
could trigger a conformational change in SecA and induce the high
affinity state through interaction with the carboxyl-terminal peptide
of SecA.
The carboxyl-terminal peptide cannot simply be the site of high
affinity binding itself, because if it were the complex would form in
solution at submicromolar concentrations, which we have shown it does
not. Presence of the carboxyl-terminal peptide is necessary for the
creation of the high affinity site, but it is not sufficient.
Interaction at other sites, which are present even in complexes between
defective variants of SecB and SecA, are required to form a complex
that then can be converted to an active form. The active form is likely
to correspond to a complex that contains SecA in a state that has high
affinity for wild-type SecB. Evidence for such a high affinity state is
provided by the demonstration that the variant forms of SecB could
compete with wild-type SecB for SecA in complex 1 but not for SecA in
complex 2. It is possible that the SecA in the complex characterized
here in solution as complex 2 in fact represents the high affinity membrane form of SecA. This SecA might well be in the conformational state that was shown by Tai and his co-workers (20) to be exchangeable with membrane-bound SecA.
It is of great interest to determine the difference between the active
and inactive complex. It seems likely the difference is one of
conformation. However, it is a nontrivial task to distinguish definitively differences in stoichiometry and mass from differences in
conformation in such a complicated system, because both components of
the complex are themselves oligomeric and the complexes continuously re-equilibrate during column chromatography. Whatever the differences are between the complexes, it is clear that SecA has at least two
regions that interact with SecB. Interactions between regions of SecA
and SecB that are as yet undefined provide sufficient energy of binding
to stabilize a complex that has a Kd value in the
micromolar range. That complex is inactive in mediating translocation
of precursors unless there is interaction between SecB and the region
at the carboxyl terminus of SecA. The region of SecB that interacts
with the carboxyl terminus of SecA includes residues from two widely
separated stretches along the primary sequence of 155 aminoacyl
residues of SecB. The residues 20, 24, 75, and 77 are likely to be in
close proximity in the quaternary structure of the SecB tetramer so
that they can interact with the carboxyl-terminal 21 residues of SecA.
We thank Angela A. Lilly for aiding in the
construction of pAL81.
*
This work was supported in part by Grants GM29798 (to
L. L. R.) and GM36415 (to C. A. K.) from the National Institutes of Health.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 in part by Biotechnology Training Grant T32-GM08336 from
the National Institute of General Medical Sciences.
¶
Current address: Ciphergen Biosystems, Inc., 490 San Antonio
Rd., Palo Alto, CA 94306.
§§
To whom correspondence should be addressed: School of Molecular
Biosciences, Washington State University, P. O. Box 644660, Pullman,
WA 99164-4660. Tel.: 509-335-6398; Fax: 509-335-9688; E-mail:
topping@wsu.edu.
Published, JBC Papers in Press, May 11, 2000, DOI 10.1074/jbc.M002885200
2
J. M. Crane and L. L. Randall,
unpublished results.
The abbreviations used are:
IPTG, isopropyl-1-thio-
Complexes between Protein Export Chaperone SecB and SecA
EVIDENCE FOR SEPARATE SITES ON SecA PROVIDING BINDING ENERGY AND
REGULATORY INTERACTIONS*
§,,¶,
,
, and§§
School of Molecular Biosciences, Washington
State University, Pullman, Washington 99164-4660 and the ** Department
of Molecular Biology and Microbiology, Tufts University School of
Medicine, Boston, Massachusetts 02111
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
host (7) harboring plasmids that
overexpress SecE (pMAN809) and SecY (pMAN510) (8) under the control of
the tac promoter. Strain HB1590 was constructed by
transformation of the same plasmids into HB1187 (MC4100,
uncB-C) (9). E. coli strain HB1732 is JM109
(New England BioLabs) harboring plasmid pAL81, which overexpresses
SecAN880, a truncated SecA consisting of the first 880 aminoacyl
residues. To construct plasmid pAL81, the SecA expression vector
pMAN400 (10) was used as template in a polymerase chain reaction with
the primer 5'-AGTCGCTGGAAGAAATGTG3-' and mutagenic primer
5'-ACCGTTGAGCTCTTATCCTACTTTGCG3-'. The mutagenic primer places a stop
codon after codon 880 of the secA gene followed by a
SacI restriction site. After MunI/SacI (MBI
Fermentas) digestion, the polymerase chain reaction product was ligated
into pMAN400 cut with the same enzymes. The secA gene was
sequenced to verify the change.
-D-galactopyranoside (IPTG)1 was added and the
cells grown 2 h longer. Cells were harvested, washed, and
suspended in a volume equal to the weight of the pellet of 50 mM Tris-Cl, pH 8, 2 mM dithiothreitol (DTT),
10% sucrose. The cell suspension was frozen by dripping into liquid
nitrogen and stored at 
70 °C. The cell suspension was thawed in
twice its volume of 30 mM Tris-Cl, pH 7.5, 2 mM
DTT, 1 mg/ml lysozyme, 1 mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride (PMSF), sonicated, and centrifuged at
178,000 × g for 1 h and 17 min (60 Ti rotor, Beckman) at 4 °C. The supernatant was loaded onto a Macroprep DEAE
(Bio-Rad) column equilibrated in 30 mM Tris-Cl, pH 7.5, 2 mM DTT. The column was washed with approximately 1 column
volume of the same buffer, and then 1 column volume of the same buffer with 0.2 M NaCl. The SecA was eluted with a gradient of 0.2 to 1.0 M NaCl in the same buffer. Fractions containing SecA
were pooled and dialyzed against 10 mM sodium phosphate, pH
6.5, 2 mM DTT at 4 °C. The dialyzed sample was applied
to a cellulose phosphate P11 (Whatman) column equilibrated in 10 mM sodium phosphate, pH 6.5, 2 mM DTT. The
column was washed with 10 mM sodium phosphate, pH 6.5, 2 mM DTT, and the SecA was eluted with a gradient of 0.01 to
0.5 M sodium phosphate, pH 6.5 in 2 mM DTT.
Fractions containing pure SecA were pooled, concentrated, dialyzed
against 10 mM HEPES (KOH), pH 7.6, 300 mM
potassium acetate (KOAc), 2 mM DTT, and stored at
70 °C.
1 cm1
for the tetramer; denatured precursor and mature galactose-binding proteins, 37,410 M1
cm1; SecA and SecAN880, 157,800 M1 cm1
for the dimer. All SecB concentrations are expressed as tetramer, and
all SecA concentrations are expressed as dimer.
DE3)pJW25 (15). 14C-SecBE77K,
and 14C-SecBL75Q were purified from strain
CK2212(BL21(DE3) secB::Tn5 srl::Tn10 recA1) containing two plasmids. One
plasmid contains the SecB variant gene under the natural promoter, and
the second plasmid contains the SecB variant gene under control of the
T7 promoter. Cells were grown in 24 ml of M9 minimal medium (14) with
0.4% glycerol and 50 µg/ml ampicillin at 30 °C. When the cell
density reached an absorbance at 560 nm of 0.6, 0.1 mM IPTG was added; 90 min later, 0.4 µCi of 14C-labeled amino
acid mixture (ICN) per milliliter of culture was added. After 10 min of
incorporation, the cells were harvested and washed with 10 mM Tris-Cl, pH 7.6, 30 mM NaCl, and the
resulting pellet was frozen at 70 °C. The pellet was thawed and
suspended in 20 mM Tris-Cl, pH 7.6, 2 mM EDTA,
0.1 mM PMSF, 100 µg/ml lysozyme and incubated on ice for
20 min. The suspended cells were sonicated and centrifuged 15 min at
16,000 × g. The supernatant was collected and
centrifuged at 356,000 × g in a TL100.1 rotor
(Beckman) for 20 min at 4 °C. The supernatant was loaded onto a
Q-Sepharose (0.8-ml bed volume; Amersham Pharmacia Biotech) column
equilibrated in 20 mM Tris-Cl, pH 7.6, 0.1 M
NaCl. The column was washed with the same buffer, and then the bound
proteins were eluted with 0.2, 0.3, 0.35, 0.4, 0.45, 0.5, and 0.6 M NaCl steps. The fractions containing SecB as determined
by SDS-polyacrylamide gel electrophoresis were pooled and concentrated
using a Centricon 30 centrifugal filter (Millipore), and the buffer was
exchanged for 10 mM HEPES (KOH) pH 7.0, 150 mM
KOAc by repeated dilution and concentration in the Centricon. The
concentration of SecB was determined by scanning a Coomassie
Blue-stained band on a SDS-polyacrylamide gel and comparing its
intensity to the intensity of bands generated from known amounts of SecB.
70 °C.
2t = 2.4 × 109
rad2/s) at 4 °C. Radioactivity in samples taken from the
total reaction mixture, the supernatant, and the suspended pellet was
quantified by liquid scintillation counting. The amount bound was
calculated as the percentage of the total radioactivity that was
recovered in the pellet after subtracting the radioactivity that
pelleted in the absence of vesicles (~3%). Recovery of radioactivity
was typically 95-100%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (42K):
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Fig. 1.
Complexes between SecB and precursor
galactose-binding protein. Upper panel, absorbance
profiles of complexes of precursor galactose-binding protein and SecB
resolved by size-exclusion chromatography. Purified unfolded
galactose-binding protein in 1.0 M GdnHCl, 10 mM Hepes (KOH), pH 7.0, 1 mM EGTA, 300 mM KOAc was diluted into a solution containing pure SecB to
final concentrations of 3 µM SecB, 3 µM
precursor galactose-binding protein, 50 mM GdnHCl, 10 mM Hepes (KOH), pH 7.6, 300 mM KOAc, 5 mM MgOAc, 1 mM EGTA, 2 mM DTT.
Immediately following mixing, the sample was analyzed by chromatography
using a TSK G3000SW column. The samples applied were mixtures of SecB
and precursor galactose-binding protein at molar ratio 3 µM SecB:3 µM precursor galactose-binding
protein as follows: wild-type SecB, dotted line; SecBE24A,
solid line; and SecBE24K, short-dashed line. SecB
only is represented by the long-dashed line and unfolded
precursor galactose-binding protein by the dotted-dashed
line. Lower panel, SDS-polyacrylamide gel
electrophoresis of trichloroacetic acid precipitates of successive
0.33-ml fractions collected during chromatography of the
SecBE24A·pGBP complex. Lane S contains 2.5% of the
quantity of sample applied. The positions of precursor
galactose-binding protein (pGBP) and SecB are
indicated.
View larger version (35K):
[in a new window]
Fig. 2.
Complexes between SecA and wild-type
SecB. Upper panel, Absorbance profiles of complexes
between SecA and SecB resolved by size-exclusion chromatography. HPLC
was carried out as described under "Experimental Procedures."
Samples applied were 4 µM SecA only, dotted
line; 4 µM SecB only, short-dashed line;
1 µM SecA:1 µM SecB, dotted-dashed
line; 4 µM SecA:4 µM SecB, solid
line; and 6 µM SecA:6 µM SecB,
long-dashed line. Lower panel, SDS-polyacrylamide
gel electrophoresis of trichloroacetic acid precipitates of successive
0.33-ml fractions collected during chromatography of the 4 µM SecA:4 µM SecB sample. Lane S
contains 0.5% of the quantity of sample applied to the column. The
positions of SecA and SecB are indicated. The relative intensity
difference of the SecA and SecB is a result of the large difference in
molecular weight of the two proteins and the fact that SecB stains less
well than does SecA.
View larger version (21K):
[in a new window]
Fig. 3.
Complexes between SecA and variants of
SecB. Absorbance profiles of complexes separated by size-exclusion
chromatography. In A: upper panel, absorbance
profiles of samples applied as follows: 4 µM SecA:4
µM SecBD20A, solid line; 4 µM
SecA:4 µM SecB, dotted line; 4 µM SecBD20A only, dashed line. Lower
panel, SDS-polyacrylamide gel electrophoresis of fractions
collected during chromatography of the SecA:SecBD20A. B,
absorbance profiles of 6 µM SecA:6 µM
SecBE24A, solid line; 6 µM SecA:6
µM SecBE24K, dashed line; 6 µM
SecA:6 µM SecB, dotted line. C,
absorbance profiles of 6 µM SecA:6 µM
SecBE77K, solid line; 6 µM SecA:6
µM SecBL75Q, dashed line; 6 µM
SecA:6 µM SecB, dotted line.
View larger version (17K):
[in a new window]
Fig. 4.
Competition by SecBL75Q for the SecA in
complex with wild-type SecB. Complexes between 4 µM
SecA and 5 µM 14C-SecB (6.8 × 107 cpm/µmol) were made in the absence (solid
line, 
) or presence (dashed line, 
) of 10 µM SecBL75Q. Each mixture was analyzed by size-exclusion
chromatography as described under "Experimental Procedures" except
that the buffer was pH 7.0 and did not contain MgOAc. The absorbance at
280 nm was monitored (dashed and solid lines),
fractions were collected by drop (approximately 0.2 ml), and the
radioactivity in each fraction was determined by liquid scintillation
counting (, ). The recovery of the radioactivity was greater than
90% of that applied.
View larger version (23K):
[in a new window]
Fig. 5.
Binding of wild-type and altered species of
SecB to membrane vesicles. The binding to inverted cytoplasmic
membrane vesicles of metabolically labeled 14C-SecB
(wild-type, L75Q, or E77K, at specific activities of 3.6 to 7.2 × 107 cpm/µmol) was determined as described under
"Experimental Procedures." The concentrations of SecB and, when
present, SecA and pGBP were 1 µM.
View larger version (12K):
[in a new window]
Fig. 6.
SecA-dependent binding of ternary
complexes as a function of SecB concentration. Various
concentrations of 14C-SecB (wild-type, L75Q, or E77K) were
assayed for binding to vesicles in both the presence and absence of 2 µM SecA. All assays included 2 µM pGBP. The
amount of SecB pelleted with vesicles in the presence of SecA minus the
amount pelleted in the absence of SecA in two experiments was averaged
for wild-type ( 
). The experiments with SecBL75Q () and SecBE77K
(
) were done once. Negative values for the amount of SecB bound
indicate that less SecB is bound in the presence of additional SecA
than in its absence. Buffer conditions in these assays were the same as
described for in vitro processing except that no ATP was
added.
View larger version (39K):
[in a new window]
Fig. 7.
Demonstration of kinetic partitioning
in vitro. Processing of pGBP was carried out immediately
after dilution of the pGBP or after a 10-min incubation at 24 °C in
the presence of 0.7 mM EGTA or 9 mM
CaCl2. After addition of SecB (lanes with
"+"), SecA, and inverted vesicles, processing was allowed to
proceed for 54 min at 37 °C. The positions of pGBP and mGBP are
indicated.
View larger version (13K):
[in a new window]
Fig. 8.
Processing of pGBP in vitro as a
function of the concentration of SecB. Processing of pGBP was
carried out for 45 min as described under "Experimental Procedures"
in the presence of wild-type SecB ( ) or SecBE77K (itrif]).
View larger version (27K):
[in a new window]
Fig. 9.
Complexes between truncated SecA and
SecB. A: upper panel, absorbance profiles of
complexes separated by size-exclusion chromatography. Samples were: 4 µM SecAN880, dashed line; 4 µM
SecA:4 µM SecB, dotted line; 4 µM SecAN880:4 µM SecB, solid
line. Lower panel, SDS-polyacrylamide gel
electrophoresis of fractions collected during chromatography of 4 µM SecAN880:4 µM SecB. B,
absorbance profiles of complexes separated by size-exclusion
chromatography. Samples were: 4 µM SecAN880:4
µM SecBL75Q, solid line; 4 µM
SecAN880:4 µM SecB, dashed line; 4 µM SecA:4 µM SecB, dotted
line.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
Current address: Combimatrix Corporation, 34935 Southeast
Douglas Ave., Suite #110, Snoqualmie, WA 98065.
Current address: Dept. of Biology, University of York, Y01 5DD
United Kingdom.
ABBREVIATIONS
-D-galactopyranoside;
DTT, dithiothreitol;
PMSF, phenylmethylsulfonyl fluoride;
KOAc, potassium
acetate;
MgOAc, magnesium acetate;
GdnHCl, guanidine hydrochloride;
pGBP and mGBP, precursor and mature galactose-binding protein,
respectively.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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