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INTRODUCTION |
In bacteria, the vast majority of extracytoplasmic proteins are
transported across the cytoplasmic membrane by the Sec apparatus (for
review, see Ref. 1). In addition to several specialized protein export
systems, another major protein export system has been discovered that,
in contrast to the Sec apparatus, is able to transport folded proteins.
This translocation pathway, denoted the twin-arginine translocation
(Tat)1 pathway, is closely
related to the
pH-dependent pathway of thylakoid membranes (reviewed in Ref. 2). Substrates for this pathway are
characterized by an essential twin-arginine motif in their signal
peptides (3, 4).
In Escherichia coli, four genes have been shown to encode
components of the Tat pathway (5-9). Three of these, tatA,
tatB, and tatC, are located in one operon,
whereas the fourth gene, tatE, is monocistronic. TatA, TatB,
and TatE are homologous proteins that are predicted to contain a single
transmembrane helix at their amino termini followed by a cytoplasmic
domain. It has been shown that TatA and TatE fulfill similar functions,
whereas TatB has a distinct role in the translocation process (8). The
fourth protein, TatC, is predicted to contain six transmembrane
helices, with the amino and carboxyl termini located at the cytoplasmic face of the membrane. TatB and TatC are essential components of the Tat
pathway (6-8), and translocation requires in addition either TatA or
TatE. However, TatA seems to be far more important than TatE, most
likely because of a greater abundance (5, 10).
Only a limited amount of information is available on the composition of
the Tat translocase. Recently, it was shown that TatA and TatB interact
with each other and are present in a large complex of ~600 kDa (11).
Notably, it was found that not all TatA was bound to TatB, suggesting
that TatA might be present in a separate complex. Thus far, it has not
been established whether TatC forms part of the 600-kDa TatA/B complex,
and the possible involvement of other components could not be excluded.
To analyze the composition of the Tat system in more detail we have
purified the complex from E. coli membranes. Here, we show
that the Tat system purifies as a TatABC complex, and we find no
evidence for the presence of additional, hitherto unidentified subunits, although the existence of further subunits cannot be excluded
because the activity of the complex has not been tested. Within the Tat
complex, TatB and TatC are the most stable components and are present
in a 1:1 ratio, suggesting a structural association. We also show that
cells containing a fusion of the TatB and TatC proteins are able to
support Tat-dependent transport, indicating that TatB and
TatC also form a functional unit and act in concert. Finally, although
some TatA is tightly associated with TatB/C, we find that the vast
majority of TatA does not co-purify with the complex, suggesting a
looser association or a separate function in the overall translocation process.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains, Plasmids, and Growth Conditions--
E.
coli strain MC4100 was the parental strain;
tatAE,
tatB,
tatC, and
tatABCDE have
been described before (5, 7, 9), and arabinose-resistant derivatives
were used as described (11). E. coli was grown aerobically
at 37 °C in TY medium (11). E. coli was grown
anaerobically in TY-GT medium, consisting of TY supplemented with
glycerol (0.5%), trimethylamine N-oxide (TMAO; 0.4%), and
ammonium molybdate (1 µM), or in minimal TMAO/glycerol medium (11). Ampicillin was used at 100 µg/ml.
The tatABC operon was amplified with the primers
AB.tatA1 (ataccATGGGTGGTATCAGTATTTG; nucleotides identical
to genomic DNA are capitalized, and restriction sites are underlined)
and AB.tatC-s (atattctagattatttttcaaactgtgggtgcgaccaattcgaTTCTTCAGTTTTTTCGCTTTCTGC; nucleotides in bold specify the Strep-tag II peptide SNWSHPQFEK; Ref. 12). The resulting product was digested with NcoI and
XbaI and cloned into plasmid pBAD24 (13), generating
pABC-s.
A vector encoding a fusion of the TatB and TatC proteins was generated
as follows. First, the tatB gene was amplified with the primers JT.B1 (gccatgccATGGCGTTTGATATCGGTTTTAGC) and
JT.B2 (gctctagagttgttgttattgttattgttgttgttgttCGGTTTATCACTCGACGA; nucleotides in bold specify a linker consisting of 10 Asn residues). The tatC gene was amplified with the primers JT.C3
(gctctagaatcgaaggtcgtTCTGTAGAAGATACTCAA; nucleotides in bold specify a factor Xa cleavage site; IEGR) and JT.C4
(ccaatgcattggttctgcagttaTTATTCTTCAGTTTTTTCG). Next,
the resulting products were digested with NcoI and
XbaI and with XbaI and PstI,
respectively, and cloned in one step into pBAD24, generating pJDT7.
To construct a vector encoding both TatA and the Tat(BC) fusion
protein, pJDT7 and pABC-s were both digested with SstII and PstI. Next, the DNA fragment encoding the Tat(BC) fusion
protein (derived from pJDT7) was cloned into the vector fragment
derived from pABC-s, thereby replacing the tatB and
tatC genes with the tat(BC) gene. The resulting
plasmid was denoted pJDT12.
To construct a vector encoding the Tat(BC) fusion protein with a
Strep-tag II peptide at its carboxyl terminus, the tat(BC) gene was amplified using the primers AB.tatB1
(ataccATGGTGTTTGATATCGGTTTTAG and AB.tatC-s (see above) and
plasmid pJDT7 as template DNA. Next, the resulting DNA fragment was
cloned into pBAD24, generating p(BC-s).
SDS-PAGE and Western Blot Analysis--
Proteins were separated
by SDS-polyacrylamide gel electrophoresis and immunoblotted and
visualized with specific antibodies (11) and horseradish peroxidase
(HRP) anti-rabbit IgG conjugates, using the ECL detection system
(Amersham Pharmacia Biotech). TatC with a Strep-tag II was visualized
directly using a Streptactin-HRP conjugate (Institut für
Bioanalytik, Göttingen, Germany).
Purification of the TatABC Complex--
Cells were grown
anaerobically in TY-GT medium to the end of the exponential growth
phase and then spheroplasted and sonicated, and the membranes were
isolated as described before (11). Membranes were solubilized in buffer
I (20 mM Tris-HCl, pH 8.0, 20% glycerol) plus 50 mM KCl and 1% digitonin (Calbiochem). To prevent
degradation of proteins, a protease-inhibitor mixture (Complete, Roche
Molecular Biochemicals) was added to buffers for spheroplasting,
membrane isolation, and membrane solubilization. Solubilized membranes were loaded on a Q-Sepharose column. The column was washed with 1 column volume buffer I containing 100 mM KCl and 0.1%
digitonin, and proteins were eluted with 2 column volumes of buffer I
containing 300 mM KCl and 0.1% digitonin. Avidin (2 µg/ml) was added to the sample to block any biotin-containing
proteins, and the sample was loaded on a 2-ml Streptactin-Sepharose
(Institut für Bioanalytik) column. The column was washed with 5 column volumes of buffer I containing 200 mM KCl and 0.1%
digitonin. Proteins were eluted with 5 × 1.5 ml of the same
buffer containing 2.5 mM desthiobiotin (Institut für
Bioanalytik). The first three fractions were pooled and concentrated to
0.5 ml using a Centricon centrifugal filter (YM-100; 100,000 molecular
weight cut-off; Amicon). This sample was loaded on a Superose 6HR gel
filtration column (Amersham Pharmacia Biotech). Elution was with buffer
I containing 200 mM KCl and 0.1% digitonin. Fractions of
0.6 ml were collected, and those containing TatABC were pooled, diluted
with buffer I to give a KCl concentration of 50 mM, and
loaded on a MonoQ column (Amersham Pharmacia Biotech). Proteins were
eluted with a linear KCl gradient (50-400 mM) in 20 column volumes.
For purification of the radiolabeled TatABC-s complex, cells were grown
in minimal medium supplemented with all amino acids except methionine
and cysteine. At the mid-exponential growth phase, the cells were
labeled with [35S]methionine for 15 min. Purification of
the radiolabeled TatABC-s complex was performed as described above.
Samples were analyzed by SDS-PAGE and fluorography, and the levels of
TatA, TatB, and TatC-s were quantified using a PhosphorImager and
ImageQuant software (Molecular Dynamics).
Isolation of Membranes and Immunoprecipitation--
Membranes
isolated from cells grown aerobically in TY medium were solubilized
with 1% digitonin, and proteins were immunoprecipitated with anti-TatA
or anti-TatB serum as described before (11). Peak fractions from the
MonoQ column of radiolabeled TatABC-s complex were immunoprecipitated
using anti-TatA, anti-TatB, or an irrelevant serum (raised against
spinach photosystem II subunit W) and visualized by SDS-PAGE and fluorography.
TMAO Reductase Activity Assay--
Cells were grown
anaerobically in TY-GT medium until the mid-exponential growth phase,
and periplasm and spheroplasts were prepared by the EDTA/lysozyme/cold
osmoshock procedure (14). Spheroplasts were lysed by sonication, and
intact cells and cellular debris were removed by centrifugation (5 min
at 10,000 × g). Membranes were separated from the
cytoplasmic fraction by centrifugation (30 min at 250,000 × g. Protein fractions were separated on a 10% nondenaturing
polyacrylamide gel, and TMAO reductase activity was visualized in the
gel using a methyl-viologen-linked TMAO reduction as described before
(15).
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RESULTS |
TatA Requires TatC for Interaction with TatB--
Previously, we
have shown TatA and TatB co-immunoprecipitate with each other and
participate in a large complex of ~600 kDa (11). However, the complex
was not purified during this work, and we did not establish whether
TatC was associated with this complex. To address these points, we
first tested whether TatC is required for interaction of TatA and TatB.
Membranes were isolated from E. coli MC4100 and the
tatAE,
tatB, and
tatC
strains, and, as shown in Fig. 1, TatA
could be immunoprecipitated from wild-type cells (lanes WT)
using the anti-TatB serum, as found previously (11). In control assays,
TatA was also immunoprecipitated from wild-type cells using an
anti-TatA serum (lane
-TatA). The immunoprecipitation of TatA using the TatB serum is not due to recognition of TatA by the anti-TatB antibodies, because TatA was not
co-immunoprecipitated in cells lacking TatB (lane
B). As expected, no immunoprecipitation is observed
in cells lacking TatA and TatE (lane
AE).
Significantly, TatA was also not co-immunoprecipitated from cells
lacking TatC (lane
C), even though these cells
contain normal levels of TatA and TatB (11), showing for the first time that TatC is involved in the complex and, moreover, indicating that
TatC is required for interaction of TatA and TatB.

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Fig. 1.
Co-immunoprecipitation of TatA using
anti-TatB serum. Membranes of E. coli MC4100
(WT), tatAE ( AE),
tatB ( B), or tatC
( C) were solubilized with digitonin, and
immunoprecipitation (IP) was performed using anti-TatB
serum. As a control, TatA was immunoprecipitated from E. coli MC4100 using anti-TatA serum. TatA was visualized by SDS-PAGE
and Western blotted using anti-TatA serum. Because anti-TatB serum
co-immunoprecipitates only a small fraction of the total TatA pool
(11), the exposure time of the film was longer for the left
panel. M, molecular mass reference marker (indicated in
kilodaltons).
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Controlled Expression of the tatABC Operon--
To analyze the
composition of the Tat complex more directly, an expression vector was
constructed encoding TatC with a Strep-tag II fusion on the carboxyl
terminus (denoted TatC-s). The Strep-tag II peptide enables detection
using Streptactin (an engineered streptavidin; Ref. 16)-HRP conjugate
and purification on Streptactin-Sepharose affinity columns. A major
advantage of the latter system is that proteins can be purified using
very mild conditions, because elution can be achieved by simply adding
low concentrations of a biotin derivative, desthiobiotin, to a
physiological buffer. Because the stability of TatB and TatC depends on
the presence of TatA and TatB, respectively (8, 11), the expression
vector (pABC-s) encoded TatA and TatB as well as TatC-s. All three
tat genes were under the control of the arabinose-inducible
PBAD promoter.
To test whether the plasmid-borne expression of TatA, TatB, and
TatC-s was fully functional, pABC-s was transformed into an E. coli strain lacking the tatABCD operon and
tatE. The latter strain is unable to grow anaerobically in
minimal glycerol/TMAO medium (5, 7, 8). Plasmid-borne
tatABC-s was able to restore this growth defect of E. coli
tatABCDE, showing that tatA,
tatB, and tatC-s are expressed and, moreover,
that the Strep-tagged derivative of TatC is functional (data not shown).
To verify the expression level of the plasmid-borne tatABC
genes, cells of E. coli strain
tatABCDE
containing plasmid pABC-s were grown in the presence of 5 or 100 µM arabinose, and TatB levels were compared with those in
wild-type cells (E. coli MC4100) by Western blotting. In the
presence of 5 µM arabinose, the expression level of TatB
is similar to that of wild-type cells (Fig.
2). In the presence of 100 µM arabinose, TatB was overproduced ~50-fold. Similar
values were obtained for the cellular levels of TatA (data not
shown).

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Fig. 2.
Expression of TatB from pABC-s. Cells of
E. coli MC4100 (WT) containing plasmid pBAD24
(vector without insert), or tatABCDE containing plasmid
pABC-s were grown in TY medium until the end of exponential growth. The
latter strain was grown in the presence of 5 or 100 µM
arabinose (lanes indicated by 5 and
100). The cells were collected by centrifugation, and TatB
was visualized by SDS-PAGE and Western blotting. To prevent
overexposure from the sample containing overexpressed TatABC, only 1 or
10% of the sample was loaded on the gel.
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Purification of a TatABC-s Complex--
To identify the proteins
that co-purify with TatC-s, digitonin-solubilized membranes (Fig.
3A, lane 1)
isolated from cells grown in the presence of 100 µM
arabinose were first subjected to Q-Sepharose chromatography. Proteins
eluting in a buffer with 300 mM KCl (Fig. 3A,
lane 2) were further purified on a Streptactin column. The
eluate from this step contains two major bands of ~27 and 31 kDa
(Fig. 3A, lane 3). Western blotting showed that these bands are TatB and TatC-s, respectively (Fig. 3, B and
C). Furthermore, a band running at ~17 kDa was visible,
which, as confirmed by Western blotting, is TatA. Finally, a number of
other bands were visible that are multimers of TatC (indicated by
C*) and a degradation product of TatB (indicated by
B*), because these bands were also detected with
Streptactin-HRP and anti-TatB, respectively (data not shown).

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Fig. 3.
Purification of TatABC-s. Membranes,
isolated from E. coli tatABCDE (pABC-s) cells
grown in the presence of 100 µM arabinose, were
solubilized, and proteins were purified using the various steps as
described under "Experimental Procedures." Fractions from the
different purification steps were analyzed by SDS-PAGE and silver
staining (A) or Western blotting using Streptactin-HRP
(B), anti-TatB serum (C), or anti-TatA serum
(D). Lane 1, solubilized membranes; lane
2, Q-Sepharose pool; lane 3, fractions from Streptactin
column concentrated in a Centricon centrifugal filter YM-100 (100,000 kDa cut-off); lane 4, peak fraction of the Superose 6HR
column; lane 5, peak fraction from MonoQ column. Loading of
lanes 1 and 2 is similar in volume, whereas
fractions 3-5 are concentrated relative to the starting volume. The
starting volume in lane 1 was 15 ml, of which 4 µl was
loaded on gel. Lane 5 contains 10 µl of a 0.5-ml fraction.
M, molecular mass reference marker (indicated in
kilodaltons).
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The TatABC proteins were further purified on a Superose 6HR gel
filtration column. TatABC eluted in a single peak corresponding to a
molecular mass of ~600 kDa (Fig. 4),
which is in agreement with the molecular mass found for the solubilized
TatA/B-containing complex in our earlier studies (11). A final
purification step was performed using anion-exchange chromatography.
Silver staining of peak fractions revealed only the presence of TatB
and TatC-s (Fig. 3A, lane 5), but Western
blotting showed that these fractions also contained TatA protein (Fig.
3D). Concentration of peak fractions using a Centricon
filter (100,000 molecular weight cut-off) and analysis by
SDS-PAGE and Coomassie staining showed a similar result: only TatB and
TatC-s were detectable; the levels of TatA are once again too low for
visualization (not shown).

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Fig. 4.
The TatABC-s complex has a molecular mass of
~600 kDa. A chromatogram of the gel filtration of the TatABC-s
complex is shown. A sample concentrated to 0.5 ml after purification of
the Tat complex on a Streptactin column was loaded on a Superose 6HR
gel filtration column, and the proteins were eluted as described under
"Experimental Procedures." Protein elution was monitored by
absorbance (A) at 280 nm. The column was calibrated with the
following proteins: thyroglobulin (669 kDa), ferritin (440 kDa),
catalase (232 kDa), and aldolase (158 kDa). MW, molecular
weight; Vo, void volume; Ve,
elution volume.
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In the experiments described above, the TatABC-s proteins were
overproduced to prepare sufficient quantities of material. However, it
was important to purify the complex from cells expressing wild-type
levels of the Tat system to test whether the complex contains any other
proteins (because these would not be overexpressed from the
tatABC plasmid). This was achieved by purifying the complex from cells grown on low levels of arabinose (5 µM), which
corresponds to wild-type levels of TatA and TatB, using the first three
steps of the purification strategy outlined above. After these
purification steps, only bands corresponding to TatB and TatC were
visible (data not shown). The presence of TatA was again confirmed by Western blotting. No other bands were detectable, strongly suggesting that the core components of the twin-arginine translocase complex are
TatA, TatB, and TatC.
The Tat Complex Contains TatB/C in a Fixed Ratio
Together with Varying Amounts of TatA--
The data shown above
indicate that TatB and TatC are easily detected in the purified
complex, but TatA is difficult to detect and quantify using these
procedures. We have found that this protein stains aberrantly with
Coomassie and very poorly with silver (not shown). We therefore used an
alternative strategy to obtain a clearer picture of the subunit ratios
in the purified complex. The Tat complex was isolated from cells grown
in the presence of [35S]methionine, and Fig.
5A shows the elution of the
radiolabeled complex from the penultimate (gel filtration) and final
ion-exchange chromatography stages. Only three bands are visible,
corresponding to TatA, TatB, and TatC-s. Using this procedure, TatA is
now clearly detected, and this is clear evidence that this subunit is
indeed an integral component of the purified complex. To analyze the subunit ratios in the various fractions, the band intensities were
quantified using a PhosphorImager. Furthermore, to be able to compare
the ratios accurately, amino-terminal sequencing was performed to
determine whether the amino-terminal methionines were present in the
TatA, TatB, and TatC proteins. The sequences obtained were MGGISI,
MFXIG, and XVEDT, respectively. The TatA and TatB sequences are exactly as predicted from the gene sequences, demonstrating that TatA and TatB do contain their amino-terminal methionines. In contrast, the tatC gene sequence predicts MSVEDT, indicating that the amino-terminal methionine is not present in the
purified TatC. Fig. 5B shows the A:B:C ratios of the lanes shown in Fig. 5A, calculated according to the number of
methionine residues in each subunit (8 in TatC, 3 in TatB, and 2 in
TatA). The figures show that the ratio of B:C is close to 1 in each
eluate, indicating that these subunits are present in equimolar
amounts. Interestingly, the ratio of A:B/C varies considerably in the
gel filtration eluate. All fractions contain at least 1 TatA per
TatB/C, but in some fractions the ratio is much greater. The peak
fractions from the final ion-exchange column contain A, B, and C in a
ratio that is very close to 1:1:1. To test how tightly TatA was bound to TatB/C, the peak fractions from the anion-exchange column were immunoprecipitated with anti-TatA (lane
A),
anti-TatB (lane
B), or an irrelevant antibody
(lane
W), and after SDS-PAGE, band intensities
were quantified using a PhosphorImager (Fig. 5C). The ratio
of B:C after immunoprecipitation with either anti-TatA or anti-TatB was
again very close to 1 (1.0 and 1.1, respectively). This again suggests
a close association of TatB and TatC. Strikingly, the ratio of A:B/C in
these two lanes is very different, being 8.8 after immunoprecipitation
with anti-TatA and 0.53 after immunoprecipitation with anti-TatB. In
conclusion, these data show that TatB and TatC remain in a fixed 1:1
ratio throughout four different chromatography stages and an extensive
immunoprecipitation procedure, strongly indicating that they are
tightly associated and might form the core of the Tat complex. In
contrast, TatA is also an integral element of the complex, but most of
this subunit is gradually lost during the purification stages, and much
of it may not even be bound to Tat(BC) at the point of membrane
solubilization. Thus, TatA seems to be more loosely associated with the
TatB/C complex.

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Fig. 5.
Subunit ratios in radiolabeled Tat
complex. The Tat complex was purified from cells grown in the
presence of [35S]methionine. A, peak fractions
eluting from the gel filtration and final anion-exchange columns. The
fraction numbers are given above the lanes. TatA,
B, and C-s are indicated together with the molecular mass markers
(lane M; indicated in kilodaltons). B, the
35S content in bands shown in A was quantitated
using a PhosphorImager, and the ratios of TatB:C (black
triangles) and TatA:C (open squares) are shown.
C, Immunoprecipitation of the peak fractions from the
anion-exchange column using anti-TatA (lane
A), anti-TatB (lane B), or
an irrelevant antibody (serum against spinach photosystem II subunit W;
lane W).
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A TatB-TatC Chimera Supports Tat-dependent
Protein Translocation--
The data described above indicate an
intimate structural link between the TatB and TatC proteins and raise
the possibility that the two subunits may act in concert. This has been
confirmed by further experiments in which we tested whether a
translational fusion between these two proteins would be functional in
E. coli cells. For this purpose, a plasmid (pJDT12) was
constructed containing the tatA gene followed by a
translational fusion of the tatB and tatC genes,
all under the control of the arabinose-inducible PBAD promoter. The tatBC gene encodes a TatBC fusion protein,
denoted Tat(BC), in which the TatB and TatC domains are separated by a spacer region of 10 asparagine residues and a factor Xa protease cleavage site (IEGR; Fig. 6). This
Tat(BC) protein comprises 443 residues and is predicted to contain
seven transmembrane helices: one transmembrane helix in the
amino-terminal domain (corresponding to the amino terminus in TatB) and
six transmembrane helices in the carboxyl-terminal domain
(corresponding to the TatC domain). Furthermore, it contains a large
cytoplasmic domain corresponding to the carboxyl-terminal domain of
TatB (including the predicted amphipathic helix), the spacer region,
and the first amino-terminal residues of the TatC protein.

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Fig. 6.
The predicted topology of the Tat(BC) fusion
protein. The topology of Tat(BC) was based upon the predicted
topologies of the individual TatB and TatC proteins (5, 7). The
open boxes represent transmembrane helices, the closed
box represents the predicted amphipathic helix in the TatB domain,
and the hatched box represents the spacer region between the
tatB and TatC domains.
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To examine expression levels of the tat(BC)
gene, cells of E. coli strain
tatABCDE
containing plasmid pJDT12 were grown in the presence of varying amounts
of arabinose, and the protein was visualized by Western blotting using
an anti-TatB serum. The Tat(BC) protein has a mobility corresponding to
its predicted molecular mass of 48 kDa and was readily detected in
samples prepared from whole cells cultured with increasing
concentrations of arabinose (Fig.
7A). However, in the absence
of arabinose it was difficult to detect the protein above background
bands in whole cells. Because we noted some toxic effects of arabinose
on some of the E. coli strains when grown on minimal
TMAO/glycerol medium, resulting in extended lag phase with increasing
concentrations of arabinose, it was important to show whether Tat(BC)
was produced also without arabinose. Moreover, the correct localization
of the Tat(BC) protein in the membrane had to be established.
Therefore, periplasmic, membrane, and cytoplasmic fractions were
prepared from E. coli
tatABCDE (pJDT12) cells
grown without arabinose and, for comparison, E. coli MC4100
cells containing vector pBAD24 (without insert). As shown in Fig.
7B, Tat(BC) could only be detected in the membrane fraction,
demonstrating that it is properly located in the membrane. The level of
Tat(BC) was substantially lower than that of TatB in wild-type cells,
but, to avoid any toxic effects of arabinose and prevent an increased
lag phase, the following experiment was performed using cells grown
without arabinose. We first tested whether the pJDT12 plasmid encoding
TatA and Tat(BC) could complement the growth defect of the E. coli
tatABCDE strain on liquid minimal TMAO/glycerol
medium. Fig. 8 shows comparisons of the
growth rates of wild-type MC4100 cells with those of
tatABCDE containing the pBAD24 vector alone or pJDT12
expressing the tatA(BC) operon. The results show that the
growth rate of E. coli
tatABCDE (pJDT12) was
very similar to that of the wild-type strain (Fig. 8), whereas the
control strain E. coli
tatABCDE (pBAD24) did
not grow at all under these conditions. These results demonstrate that,
despite the low levels of Tat(BC) as compared with TatB in wild-type
cells, Tat(BC) is able to restore the growth defect of a strain lacking both TatB and TatC. This shows not only that both TatB and TatC domains
are functional individually but that the Tat(BC) fusion protein is also
functional as a unit.

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Fig. 7.
Expression of the tat(BC)
gene. A, cells of E. coli
tatABCDE (pJDT12) were grown in TY medium until the end
of exponential growth in the presence of various concentrations of
arabinose (indicated in micromolars). Cells were collected by
centrifugation, and Tat(BC) was visualized by SDS-PAGE and Western
blotting using anti-TatB serum. B, cells of E. coli MC4100 (pBAD24) and tatABCDE (pJDT12) grown the
absence of arabinose were collected by centrifugation, and periplasmic,
membrane, and cytoplasmic fractions were isolated. TatB and Tat(BC)
were visualized by SDS-PAGE and Western blotting. TatB and Tat(BC) are
indicated together with the molecular mass markers (indicated in
kilodaltons).
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Fig. 8.
The Tat(BC) chimera supports anaerobic growth
in minimal TMAO/glycerol medium. Overnight cultures of E. coli MC4100 (pBAD24) (indicated with squares),
tatABCDE (pJDT12) (indicated with triangles),
or tatABCDE (pBAD24) (indicated with circles)
were diluted 100-fold in fresh minimal TMAO/glycerol medium and
incubated at 37 °C. Growth was determined by optical density
readings at 600 nm.
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The periplasmic enzyme TMAO reductase (TorA), which is required for
growth on minimal TMAO/glycerol medium, is a Tat-dependent substrate (5, 17). Therefore, a final test on the functionality of the
Tat(BC) fusion protein was performed by analyzing whether active TMAO
reductase was present in the periplasm, using a methyl-viologen-linked TMAO reduction assay on a nondenaturing gel (Fig. 9). The results show
that, in wild-type cells containing pBAD24, TMAO reductase activity is
found in the periplasm as expected. Substantial activity is also found
in the cytoplasm because not all of the TMAO reductase is exported
under these conditions, as found in other studies (17). No periplasmic
activity is detected in the
tatABCDE cells containing
this vector, again as expected because the export of this protein is
completely dependent on the Tat pathway (5, 17). Significantly, TMAO
reductase is found in the periplasm of
tatABCDE cells
expressing the tatA(BC) operon (Fig.
9, lanes pJDT12)
confirming that the Tat pathway is operational. The export efficiency
is low in cells grown without arabinose, but upon induction by
arabinose the export efficiency is clearly increased to the point where
export efficiency is comparable with that found in wild-type cells. In
conclusion, our data show that the Tat(BC) fusion protein is active in
cells lacking TatB and TatC, supporting the hypothesis that TatB and
TatC form a functional unit within the Tat complex.

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Fig. 9.
Export of active TMAO reductase in cells
expressing a tatA(BC) operon. Cells of MC4100
(pBAD24) or E. coli tatABCDE containing pJDT12
(encoding TatA and Tat(BC)) were grown anaerobically in TY-GT medium
until the mid-exponential growth phase. Cells of the latter strain were
grown in the presence of various concentrations of arabinose (indicated
in micromolars). E. coli tatABCDE (pBAD24) was
also analyzed, but because this strain is not able to grow
anaerobically, these cells were grown in the same medium under aerobic
conditions. Next, cells were collected by centrifugation and
fractionated, and TMAO reductase (TorA) activity in the periplasm
(p), membrane (m), or cytoplasm (c)
was visualized by analysis on a nondenaturing polyacrylamide gel and
activity staining. 5 µg of protein was loaded in each lane.
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The Tat(BC-s) Chimera Has a Molecular Mass of ~600
kDa--
To test whether the Tat(BC) chimera forms large molecular
mass complexes on its own, the Tat(BC) chimera was modified to contain a carboxyl-terminal Strep tag (as added to wild-type TatC in the purification work described above). Addition of the Strep tag does not
affect activity because the chimera is active in
tatB and
tatC cells (not shown). The construct was expressed in
the absence of TatA (in
tatABCDE cells), and Tat(BC-s)
was purified using the first three steps of the purification protocol
as described for the TatABC-s complex. The results of the last gel
filtration step are shown in Fig. 10.
The Tat(BC-s) protein elutes in a peak corresponding to a molecular
mass of ~600 kDa, demonstrating that the Tat(BC-s) protein forms a
large complex even in the absence of TatA.

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Fig. 10.
The Tat(BC-s) chimera has a molecular mass
of ~600 kDa. E. coli tatABCDE cells
containing p(BC-s) were grown aerobically in TY medium, and the
Tat(BC-s) protein was partially purified using the same protocol as
described for the TatABC-s complex. Only the results of the third step,
i.e. purification on the Superose 6HR gel filtration column,
are shown. The TatBC-s protein in each fraction was analyzed by
SDS-PAGE and Western blotting using antibodies to TatB. The elution
profiles of molecular mass markers (see legend to Fig. 4) are
indicated.
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DISCUSSION |
In the present study we have purified a complex from
E. coli containing all three of the major known Tat
components, namely TatA, TatB, and TatC. We find no evidence for the
presence of novel membrane-bound proteins that would represent the
products of hitherto unidentified tat genes. We emphasize,
however, that the purified complex has not been shown to be active in
an in vitro assay, and we cannot therefore rule out the
possibility that further subunits may remain to be identified.
An important point to emerge from this study is that TatB and TatC are
clearly present in a fixed 1:1 ratio, suggesting a close structural
association between these subunits. This suggestion is reinforced by
the finding that cells lacking tatB and tatC genes but containing instead a single polypeptide in which TatB and
TatC are fused are capable of Tat-dependent protein
translocation. This was demonstrated by the ability of these cells to
grow by anaerobic respiration on TMAO and their ability to export
active TMAO reductase into the periplasm, even when expression levels of the Tat(BC) chimera are low. These results show that TatB and TatC
form a functional unit within the Tat complex, and we conclude from
these data that TatB and TatC must act in concert.
These data have implications for the Tat translocation mechanism. At
present we do not know which subunits act as either the initial
receptor or as the translocation channel. However, our data indicate
that TatB and TatC are likely to carry out one or more particular
functions together, and this in turn suggests that TatA may serve a
different function. This idea is supported by the observation that the
amount of the third component of the translocase, TatA, varies much
more in the purification steps. This may be significant in terms of
the translocation mechanism. One possibility is that the "core"
TatB/C complex contains low amounts of TatA and that additional
molecules are recruited to form the full, active complex. The stably
bound TatA molecules might, for example, serve as a nucleation point
for the binding of further TatA molecules. We have shown before that
only a minor fraction of the total TatA pool is in a complex with TatB
(11), and this study has confirmed the following point: the majority of
the Tat(BC) is efficiently bound by the affinity column, but the vast
majority of the TatA is not. However, it is important to stress that
the fraction of TatA that is in a complex with Tat(BC) is fairly
tightly bound, because TatA was still present after purification using
a variety of columns and an immunoprecipitation step with anti-TatB
serum. Co-immunoprecipitation experiments furthermore demonstrated that
TatC is required for the co-immunoprecipitation of TatA using anti-TatB
serum, suggesting that TatA and TatB may not interact directly, but
only through TatC (although this point remains to be tested more
directly, because TatC may simply affect the conformations of TatA or TatB).
The apparent molecular mass of the purified Tat complex is ~600 kDa,
indicating that multiple TatA, B, and C subunits must be present. An
exact calculation of the number of subunits is difficult to make,
because the molecular mass determined is that of the TatABC complex in
digitonin micelles, and the detergent could contribute to the size
estimation. Thus, for an accurate measurement of the Tat complex, other
methods must be used. Further studies are also required to characterize
the complex in other respects. First, it has to be established whether
the purified complex is active in a reconstituted system, because this
would help to determine whether the TatABC proteins are indeed the only components required for Tat-dependent transport and whether
the unbound TatA subunits must be recruited for activity. Finally, it
is presently completely unclear how the Tat complex is able to
transport large folded proteins without compromising membrane impermeability. Structural analysis of the purified complex is clearly
required for a more detailed understanding of its organization and mechanism.