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Evidenceforinteractionsbetweendomainsof TatA
and TatBfrommutagenesisoftheTatABC subunits
of thetwin-arginine translocase
Claire M. L. Barrett and Colin Robinson
Department of Biological Sciences, University of Warwick, Coventry, UK
The twin-arginine translocation (Tat) system operates
in the plasma membranes of a wide range of bacteria
as well as the thylakoid membrane in plant chloro-
plasts (reviewed in [1,2]). Working in parallel with the
Sec system, it is responsible forthe export of a subset
of proteins into the periplasm, outer membrane or
extracellular medium, andthe primary defining attrib-
ute ofthe system is its ability to transport proteins in
a fully folded state [3,4]. Particular attention has
centred on a series of periplasmic proteins that are
exported only after binding redox cofactors such as
FeS or molybdopterin centres [5–8] although it should
also be emphasized that the system also transports
proteins that do not bind cofactors [1,2].
Substrates forthe Tat pathway are exported post-
translationally [8] after synthesis with cleavable, N-ter-
minal signal peptides that almost invariably contain an
essential twin-arginine motif in the N-terminal domain
[9,10]. They then interact with a translocon in the
inner membrane that consists, minimally, of three sub-
units (TatABC) in Escherichia coli and several other
Gram-negative bacteria studied to date. Genetic stud-
ies indicate that thetatABC genes are all important
for Tat activity although a fourth gene, tatE, encodes
Keywords
green fluorescent protein (GFP); Tat system;
twin-arginine; protein transport; signal
peptide
Correspondence
C. Robinson, Department of Biological
Sciences, University of Warwick, Coventry,
CV4 7AL, UK
Fax: +44 2476523701
Tel: +44 2476523557
E-mail: Crobinson@bio.warwick.ac.uk
(Received 13 December 2004, revised 25
February 2005, accepted 8 March 2005)
doi:10.1111/j.1742-4658.2005.04654.x
The twin-arginine translocation (Tat) system transports folded proteins
across the bacterial plasma membrane. Three subunits, TatA, B and C, are
known to be involved but their modes of action are poorly understood, as
are the inter-subunit interactions occurring within Tat complexes. We have
generated mutations in the single transmembrane (TM) spans ofTatA and
TatB, with the aim of generating structural distortions. We show that sub-
stitution in TatBof three residues by glycine, or a single residue by proline,
has no detectable effect on translocation, whereas the presence of three gly-
cines in theTatA TM span completely blocks Tat translocation activity.
The results show that the integrity oftheTatA TM span is vital for Tat
activity, whereas that ofTatB can accommodate large-scale distortions.
Near-complete restoration of activity in TatA mutants is achieved by the
simultaneous presence of a V12P mutation in theTatB TM span, strongly
implying a direct functional interaction betweentheTatA ⁄ B TM spans.
We also analyzed the predicted amphipathic regions in TatAandTatB and
again find evidenceof direct interaction; benign mutations in either subunit
completely blocked translocation of two Tat substrates when present in
combination. Finally, we have re-examined the effects of previously ana-
lyzed TatABC mutations under conditions of high translocation activity.
Among numerous TatA or TatB mutations tested, TatA F39A alone
blocked translocation, and only substitutions of P48 and F94 in TatC
blocked translocation activity.
Abbreviations
GFP, green fluorescent protein; Tat, twin-arginine translocation; TM, transmembrane; TMAO, trimethylamine N-oxide; TorA, TMAO
reductase.
FEBS Journal 272 (2005) 2261–2275 ª 2005 FEBS 2261
a TatA paralog of minor importance in some species
[7,8,11–13]. Only two tat genes (designated tatC and
tatA) are thought to be important in some Gram-
positive species, with a single gene product apparently
fulfilling both TatAandTatB functions [14–16].
Studies on the Tat mechanism are at an early stage.
The Tat subunits are not related to any proteins in the
database and most studies point to a mechanism that
is unique among known protein transport systems.
However, recent studies have begun to unravel some
salient features of this system. Protein expression ⁄ puri-
fication approaches have resulted in the characteriza-
tion of two distinct complexes in E. coli: a TatABC
complex and homo-oligomeric TatA complex. The
TatABC complex has a mass of 600 kDa in deter-
gent and contains multiple copies of TatABC; within
this complex, TatBand TatC are in stoichiometric
amounts andthe two subunits appear to function as a
unit [17]. Approximately equal numbers ofTatA sub-
units are present [17,18] but the vast majority of TatA
is found as separate, apparently homo-oligomeric com-
plexes [17,19,20]. In vitro cross-linking studies on the
plant thylakoid [21] or E. coli Tat system [22] have
shown that substrates initially bind to theTatB and
TatC subunits, and it thus appears that these subunits
cooperate to form the substrate binding site. In plants,
the TatA homolog was only found to cross-link to
the Hcf106 ⁄ cpTatC complex (corresponding to bacterial
TatBC) in the presence of substrate and a proton
motive force [23]. On the basis of these studies, it has
been proposed that binding of substrate to the TatBC
subunits triggers the recruitment ofthe separate TatA
complex to form an active translocation system.
In an effort to pinpoint important regions ofthe Tat
subunits, the three proteins have been subjected to
site-specific mutagenesisand a number of key regions
or residues have been identified [24–27]. TatA and
TatB are single-span proteins with C-terminal, cyto-
plasmic domainsand each has also been truncated
from the C-terminus in order to delineate the regions
important for activity [28]. Site-specific mutagenesis
has also been used to assess the importance of residues
in the predicted amphipathic domainsand cytoplasmic
regions ofTatAand TatB, andthe highly conserved
residues of TatC have also been probed [24–27]. In this
report we have analyzed the transmembrane regions of
TatA and TatB, in an effort to analyze their import-
ance for Tat function. We show that mutations
designed to destabilize theTatB TM span through sub-
stitution by proline or multiple glycine residues have
no detectable effect, whereas some ofthe TatA
mutants are severely affected or blocked in transloca-
tion activity. We also present evidencefor interactions
between the TM spans ofTatAand TatB, and
between the amphipathic regions. Finally, we have
re-examined the numerous mutations made previously
in TatA, B and C and we present new information on
potentially important TatAand TatC mutants.
Results
Analysis ofTatAandTatB mutants
The overall structures oftheTatAandTatB subunits
are similar: both contain a single TM span, with very
short periplasmic N-terminal regions and cytoplasmic
domains that are relatively small in the case of TatA
( 40 residues) and larger in TatB ( 90 residues).
The TM spans and cytoplasmic domains are separated
by regions that are strongly predicted to form amphi-
pathic a-helices [19,20]. In the present study we have
generated mutations in the TM and amphipathic
regions ofTatAandTatB (see below) in order to
probe the importance of this region, especially with
respect to a possible role forTatA as the translocation
channel. In order to present a comprehensive analysis
we have analyzed in parallel the translocation activity
of TatABC mutants described in several previous stud-
ies [24–27]. This was considered important because one
of the mutants exhibited unexpected properties when
compared with previous findings.
The effects ofthe mutations were analyzed using
two types of export assay. The first involves expression
of the mutated tatABC operon in the arabinose-indu-
cible pBAD24 vector in a tat null background (Dtat-
ABCDE strain). The cells were fractionated and the
distribution of a known Tat substrate, trimethylamine
N-oxide (TMAO) reductase (TorA) was analysed using
a native gel activity assay. This assay is not quantita-
tive but defects in translocation are usually apparent
through an increased accumulation of TorA in the
cytoplasm. It should be noted that this vector expres-
ses thetatABC operon by a factor of 10–20 fold
higher compared with wild-type TatABC levels. This
means that minor, and even moderate defects in trans-
location activity may not be revealed because the
higher levels of Tat apparatus might be able to com-
pensate for defects. Moreover, this assay is qualitative
rather than quantitative because the appearance of the
TorA signal in the native gels is not linear with time.
In summary, this assay is best suited for identification
of major defects in translocation activity.
The second assay involves synthesis of a construct
comprising the presequence of TorA linked to green
fluorescent protein (GFP), which is efficiently exported
by the Tat pathway under these conditions [24,26,28].
Mutagenesis ofTatABC C. M. L. Barrett and C. Robinson
2262 FEBS Journal 272 (2005) 2261–2275 ª 2005 FEBS
In these experiments, synthesis of TorA-GFP was
induced for 2 h using the pBAD vector, after which
the arabinose was removed and IPTG was added to
induce expression oftatABCfromthe compatible
pEXT22 plasmid (although this plasmid is relatively
leaky, thus TatABC are synthesized at appreciable
rates throughout the growth ofthe cells). Membranes
were isolated after a 3 h induction with IPTG. This
assay is effectively semiquantitative (as the cells are
again analyzed over a relatively long period) but is
more reproducible than the TorA export assay and the
pEXT22 plasmid produces TatABC at lower levels (we
estimate approximately three- to fivefold more than
wild-type [28]). Most ofthe data presented below
involve the use of this assay but it should be empha-
sized that all mutants were tested several times using
the both types of export assay. TorA export data are
shown only where there were minor discrepancies with
the TorA-GFP data.
The experimental system varied from those of previ-
ous studies [24,26] in important respects. We recently
found [28] that the Tat system is inhibited by the pres-
ence of arabinose (for unknown reasons) and TorA
export assays were conducted in a slightly different
manner compared to previous studies: only 50 lm ara-
binose was used for induction (instead of 200 lm).
With the TorA-GFP export assays, TorA-GFP was
induced with arabinose for 2 h, after which the arabi-
nose was removed andthe cells incubated with IPTG
for 3 h to induce expression ofthe mutated tatABC
operons. We have found that these conditions give
more reproducible results andthe Tat pathway of
wild-type cells is shown below to be highly active at
the time of analysis.
First we analyzed TatABC levels in cells expressing
the various mutated subunits, andthe data for the
TatA andTatB mutants (expressed using the pBAD
vector) are shown in Fig. 1. The expression of the
wild-type tatABCfromthe pBAD-ABC is illustrated
in the indicated lane, with wild-type cells in the adja-
cent MC4100 lane; it is evident that theTatA and
TatB proteins are produced from pBAD-ABC at eleva-
ted levels as described above. No TatC signal is evi-
dent in wild-type cells because this protein is detected
using antibodies to the Strep-tag II on the TatC sub-
unit in the pBAD-ABC vector. In the other control
lane, no Tat components are detected using mem-
branes from DtatABCDE cells (denoted DABCDE in
Fig. 1 and other figures) as expected. The remaining
lanes contain membrane samples from DtatABCDE
cells expressing pBAD-ABC in which mutations are
present in theTatA or TatB subunit as indicated.
Fig. 1. TatABC expression levels in cells expressing wild-type or mutated TatA ⁄ B subunits. Membranes were isolated from wild-type
MC4100 cells, DtatABCDE cells (DABCDE)andDtatABCDE cells expressing pBAD-ABC containing mutations in theTatA or TatB subunit as
indicated. Samples were immunoblotted using antibodies to TatA, TatB or the Strep-tag II on TatC. Asterisks denote strains in which a
Strep-tag II is not detected by immunoblotting.
C. M. L. Barrett and C. Robinson Mutagenesisof TatABC
FEBS Journal 272 (2005) 2261–2275 ª 2005 FEBS 2263
Although the expression levels vary to some extent, all
of the mutant proteins are present at similar levels,
with the possible exception ofTatA ⁄ G2A which exhib-
its a relatively low TatB signal for unknown reasons.
One anomaly is, however, evident with two newly gen-
erated TatB mutants, E8Q and L63A: TatAand TatB
are formed at typical levels but the TatC signal is com-
pletely absent (lanes denoted by asterisks). This is
reproducible and, because these mutants display high
levels of Tat activity (see below), we assume that the
C-terminal Strep-tag II has been removed from the
proteins after expression. Some ofthe other mutants
also have this property (see below).
Although the primary aim was to analyze new
mutants affected in the amphipathic or TM regions,
previously analysed TatA mutants containing single
amino acid changes were also analyzed in terms of
their ability to export TorA and TorA-GFP, and the
data fortheTatA mutants are shown in Fig. 2. With
the TorA export assays in Fig. 2A, it is observed that
the bulk ofthe activity is found in the periplasm in
wild-type cells and cells expressing pBAD-ABC, as
expected (lanes P) with very little cytoplasmic signal
evident. TorA is found exclusively in the cytoplasm in
DtatABCDE cells, where it migrates more slowly in the
gel system (denoted by an asterisk). TheTatA mutants
all export TorA with high efficiency with the exception
of F39A, where all ofthe TorA is present as the cyto-
plasmic form. Some cytoplasmic TorA is also evident
with L25A.
The TorA-GFP export assays are in good agreement
with the TorA data (Fig. 2B). In pEXT-ABC-expres-
sing cells, the bulk of GFP is found as mature-size
protein in the periplasm (P), whereas GFP is found
only in the cytoplasm and membrane fractions (C, F)
in cells expressing the pEXT22 vector. Some of this
protein is present as precursor form (TorA-GFP) and
some mature-size GFP is also present, presumably
due to proteolytic clipping. No signal is observed in
DtatABCDE cells that do not synthesize TorA-GFP (a
control forthe specificity ofthe GFP antibodies). All
of theTatA mutants export TorA-GFP with high effi-
ciency except F39A, which is again completely defect-
ive in translocation. Whereas some cytoplasmic TorA
A
B
C
Fig. 2. TheTatA F39A mutant is inactive whereas other TatA ⁄ B
mutants show no detectable loss of translocation activity. (A) Dtat-
ABCDE cells expressing pBAD-ABC or the same vector containing
mutations in tatA were induced using 50 l
M arabinose for 3.5 h,
and cytoplasmic (C) and periplasmic fractions (P) were prepared as
detailed in Experimental procedures. These fractions were electro-
phoresed on native polyacrylamide gels that were subsequently
stained for TMAO reductase (TorA) activity. Asterisk denotes
slower-migrating cytoplasmic form of TorA. (B) A TorA-GFP con-
struct was expressed in DtatABCDE cells using an arabinose-
inducible vector for 2 h. The arabinose was then washed out and
wild-type or tatABC operons containing the same tatA mutations as
in (A) were expressed for 3 h using the isopropyl thio-b-
D-galacto-
side-inducible pEXT22 plasmid as detailed in Experimental proce-
dures (pEXT-ABC plasmid contains wild-type tatABC operon).
Cytoplasmic, membrane and periplasmic fractions (C, M, P), were
isolated and immunoblotted using antibodies to GFP. The mobility
of mature-size GFP is indicated. (C) TatB mutants described in
Fig. 1 were analyzed for export of TorA-GFP (B) exactly as des-
cribed in (B) forTatA mutants.
Mutagenesis ofTatABC C. M. L. Barrett and C. Robinson
2264 FEBS Journal 272 (2005) 2261–2275 ª 2005 FEBS
was evident with the L25A mutation, as described
above, no defect is apparent using TorA-GFP as a
substrate. Because signal strengths are not linearly
related to protein activity in the native gel TorA assay,
we are more inclined to regard the TorA-GFP data as
evidence of high translocation activity, although other
possibilities can not be excluded.
Similar tests on theTatB mutants are shown in
Fig. 2C. The results show that all ofthe strains effi-
ciently export TorA-GFP, including the E8Q and
L63A mutants that exhibited no signal with the Strep-
tag II immunoblots for TatC in Fig. 1.
Inhibitory effects of mutations in the predicted
amphipathic regions ofTatAand TatB
Considerable attention has centred on possible roles of
conserved predicted amphipathic regions that are
highly conserved in both TatAand TatB. These
regions effectively bridge the transmembrane helices
and soluble cytoplasmic domains, and truncation ana-
lysis [29] has shown that they are essential for translo-
cation activity. Their sequences are shown in Fig. 3A.
In a previous report [24], we analyzed the effects of
changing three lysine residues in TatA (residues 37, 40
and 41) to glutamine, and in a second mutant we addi-
tionally changed K24 to alanine. These mutants,
denoted TatA ⁄ )3K andTatA ⁄ )4K previously [24]
were shown to be active albeit with reduced efficien-
cies. These TatA mutants have been re-made (and re-
named TatA ⁄ 3K > Q andTatA ⁄ K24A,3K > Q) after
finding several revertants in recent studies of previ-
ously analyzed mutants. In the case of TatB, it was
previously found that changing two arginines (residues
37 and 40) to asparagine (TatB ⁄ 2R > N), or three
lysines (residues 65, 67 and 68) to glutamine
(TatB ⁄ 3K > Q) had little effect on the efficiency of
Tat-dependent export [24]. In the present report we
have re-assessed these mutants and another mutant
combining the two sets of mutations in TatB
(TatB ⁄ 2R > N,3K > Q). This new mutant is indica-
ted by ‘Ù’ in Fig. 3. The expression profiles are shown
in Fig. 3B, which shows TatAandTatB to be synthes-
ized in all cases, although again at slightly varying lev-
els. Strep-tagged TatC is formed in every case except
TatA ⁄ K24A,3K > Q, but because this mutant is act-
ive (see below) we again believe that the TatC protein
is present but lacking the Strep-tag II.
Export assays using these mutants are shown in
Fig. 4A. The data show that all three TatB mutants
TatA
TatA
TatB
TatC
TatA
TatB
TatC
pBAD-ABCs
pBAD-ABCs
2R>N
K24A
K24A
K24A
K24A
K24A
3K>Q
3K>Q
3K>Q
3K>Q
3K>Q
3K>Q/
2R>N
2R>N
3K>Q
3K>Q
3K>Q
3K>Q
2R>N 3K>Q
2R>N 3K>Q
∆ABCDE
∆ABCDE
TatB
16
25
30
*
A
BC
Fig. 3. Mutations in the predicted amphi-
pathic regions ofTatAand TatB. (A) Primary
sequences ofthe amphipathic regions with
the targeted residues indicated by arrows
and numbered. The changes introduced in
the various mutants are indicated and under-
lined. (B) pBAD-ABC cells, DtatABCDE cells
and DtatABCDE cells expressing pBAD-ABC
containing the ‘amphipathic’ mutations
shown in (A) were analyzed by immunoblot-
ting with antibodies to TatA, TatBand the
Strep-tag II on TatC. Asterisks denote
strains that exhibit no signal with Strep-tag
II antibodies. (C) as in (B), except with
strains expressing combinations of muta-
tions in TatA + TatB. ‘^’ denotes new muta-
tions analyzed in this study. Mutations in
TatA are shown underlined and in grey font.
Mobilities of molecular mass markers are
given on the left.
C. M. L. Barrett and C. Robinson Mutagenesisof TatABC
FEBS Journal 272 (2005) 2261–2275 ª 2005 FEBS 2265
exhibit efficient export of TorA-GFP, in that the vast
majority of protein is found in the periplasmic fraction.
This includes the new TatB mutant (TatB ⁄ 2R > N,
3K > Q) in which five basic residues are substituted;
surprisingly, there is little evident effect. Among the
TatA mutants, TatA ⁄ 3K > Q was previously described
as active [24] but Fig. 4A shows this not to be the case
with the newly generated mutant, which fails to export
TorA-GFP to any detectable extent. No export of TorA
was observed either (data not shown). Surprisingly, the
presence of an additional mutation (K24A) enables
export to take place, with theTatA ⁄ K24A,3K > Q
quadruple mutant exhibiting moderate export activity.
Some precursor form of TorA-GFP is present in the
cytoplasm and ⁄ or membrane fractions but considerable
amounts of periplasmic TorA and GFP are present. It is
likely that the original TatA ⁄ 3K > Q mutant [24] simi-
larly acquired an additional mutation prior to analysis
that enabled translocation to occur, although this has
yet to be confirmed. Other TatABC mutations described
previously [24,26] have been remade and shown to have
unchanged properties.
We also expressed tatABC operons in which these
multiple mutations in the amphipathic regions were
combined; in the relevant Figures theTatA mutations
are shown underlined for simplicity. We previously
reported data [24] on two such combinations:
(TatA ⁄ K24A, 3K > Q + TatB ⁄ 3K > Q) and (TatA ⁄
3K > Q + TatB ⁄ 2R > N), and both mutants were
described as active. In this report we have reassessed
the effects of these mutations (as the former TatA
mutant TatA ⁄ 3K > Q is now known to be inactive on
its own, as shown above) and have constructed several
new permutations as detailed in Fig. 3. These new
mutations are again denoted by ‘Ù’. Figure 3C shows
that strains synthesizing all ofthe multiple TatAB
mutations contain TatABC at similar levels and activ-
ity assays are shown in Fig. 4(B,C).
These ‘mixed amphipathic’ mutations exhibit very
interesting properties. Figures 2 and 4A showed that
the TatA ⁄ K24A andTatB ⁄ 2R > N mutations support
wild-type levels of export activity but Fig. 4B shows
that the combined (TatA ⁄ K24A + TatB ⁄ 2R > N)
mutations severely disrupt activity, with no periplasmic
A
B
C
Fig. 4. Combinations of mutations in the
TatA ⁄ TatB amphipathic regions have partic-
ularly severe effects on translocation activ-
ity. (A) Mutants containing alterations in the
predicted amphipathic regions of either TatA
or TatB (as detailed in Fig. 3) were analyzed
for export of TorA-GFP using the assay pro-
tocols detailed in Fig. 2. For clarity, muta-
tions in TatA are shown underlined and in
grey. (B) Combinations of mutations in the
amphipathic regions ofTatAand TatB,
whose structures and expression profiles
are illustrated in Fig. 3, were tested for the
export of TorA-GFP. Mutations in TatA are
shown underlined and in grey font; addition-
ally, labels on the right indicate whether
mutations are in TatA or TatB. (C) Dtat-
ABCDE cells, or DtatABCDE expressing
pBAD-ABC or the same vector containing
mutations in both TatAandTatB as indica-
ted, were assayed for export of TorA using
the protocol described in Fig. 2.
Mutagenesis ofTatABC C. M. L. Barrett and C. Robinson
2266 FEBS Journal 272 (2005) 2261–2275 ª 2005 FEBS
mature-size GFP detected at all after synthesis of
TorA-GFP. Note that mature-size GFP accumulates in
the cytoplasm, and not the full precursor protein; this
is due to proteolytic clipping ofthe signal peptide
when export is blocked [28,30]. Figure 4C shows TorA
export assays in which the vast majority of TorA is
found in the cytoplasm. A minor fraction of TorA
is found in the periplasm, indicating that the mutant
is not completed blocked in Tat-dependent transloca-
tion, but this mutant is clearly badly compromised. An
almost identical result is obtained with (TatA ⁄ K24A
+ TatB ⁄ 3K > Q), and it should again be noted that
the individual TatAandTatB mutants show no appar-
ent defects. These data show that the mutations have
synergistic effects andthe particularly severe effects on
TorA-GFP export raise the intriguing possibility that
these mutations somehow affect the export of GFP
differently when compared to TorA.
The next two mutants in Fig. 4B (from left to right)
contain TatA ⁄ 3K > Q in combination with either
TatB ⁄ 2R > N or TatB ⁄ 3K > Q. Both combinations
exhibit no detectable Tat activity and this is unsurpris-
ing as we showed above that the ‘new’ TatA ⁄ 3K > Q
mutant is inactive on its own. Ofthe remaining
mutants (TatA ⁄ K24A, 3K > Q + TatB ⁄ 3K > Q) is
completely inactive, although the individual TatA and
TatB mutants did exhibit activity, andthe final mutant
containing five changes in TatA plus four in TatB is
likewise totally inactive. In all, these mutants empha-
size the importance ofthe amphipathic region but they
also show forthe first time that combinations of
TatA ⁄ TatB mutations can have far more dramatic
effects than the individual mutations.
Mutations in the transmembrane spans
of TatAand TatB
Deletion ofthe TM spans ofTatA or TatB leads to a
loss of activity [31] but the important characteristics of
these regions have not been probed. We constructed
a series of new TatA ⁄ B mutants containing changes
within the TM spans, for two reasons. First, it has
been suggested that TatA may form the translocation
channel, in which case drastic structural alterations
may be expected to selectively block the translocation
event. Secondly, mutations affecting the structure and
orientation ofthe TM span may be expected to disrupt
the interactions with other Tat components, and this
would provide information on the inter-subunit associ-
ations occurring within andbetween Tat complexes.
Both of these areas are poorly understood at present.
It has been shown with other proteins that the intro-
duction of proline residues has a marked effect on the
structures of TM spans [32,33], usually introducing dis-
tortions of major proportions, and such substitutions
were made in the TM spans ofTatAandTatB in the
present study. We also substituted three residues in the
TatA andTatB TM spans by glycine. The presence
of glycine can also lead to increased flexibility in TM
helices [34]. However, glycine residues can also play
important roles in modulating inter-helix interactions
[35] andthe effects of inserting or removing these resi-
dues may therefore be less predictable than with pro-
line mutations. Nevertheless, the presence of three
consecutive glycines should lead to a significant struc-
tural effect in either case. The proline and glycine sub-
stitutions were made near the centre ofthe TM span
in order to maximize possible structural effects.
Figure 5A shows the sequences oftheTatA and
TatB TM spans, together those ofthe mutated forms.
With TatA, residues 11–13 were changed to glycine in
one case (TatA ⁄ 3Gly) and a single residue was chan-
ged to proline in the centre ofthe TM span in another
(TatA ⁄ I12P). We also changed three additional
residues to proline in theTatA ⁄ 3Gly mutant (TatA ⁄
3Pro3Gly) and then a further three residues to glycine
in the same subunit (TatA ⁄ 3Pro6Gly). With TatB, resi-
dues 11–13 were changed to glycine (TatB ⁄ 3Gly) or a
single residue to proline (TatB ⁄ V12P).
The expression characteristics of these mutants are
shown in Fig. 5B. With the simplest TatA mutants,
TatA ⁄ 3Gly andTatA ⁄ I12P, TatABC are all present
at expected levels. With TatA ⁄ 3Pro3Gly, TatA and
TatB are present at the usual levels but TatC is not
detected at all. However, as this mutant is highly act-
ive (see below) it appears that this is another example
of the C-terminal Strep-tag II being clipped or modi-
fied. With the most drastic oftheTatA mutants,
TatA ⁄ 3Pro6Gly, TatBand TatC are synthesized but
no TatA is detected. Fractionation of cells synthes-
izing TatA ⁄ 3Pro3Gly shows the presence of full-
length protein in the cytosol, consistent with a slight
defect in membrane-insertion; in contrast, smaller
fragments oftheTatA ⁄ 3Gly6Pro protein are found in
the cytosol (data not shown). This suggests that the
TatA ⁄ 3Gly6Pro protein is degraded either within the
membrane or, perhaps more likely, after failure to
insert into the membrane.
Cells expressing the simpler ofthetatB mutants,
TatB ⁄ V12P, appear to contain TatABC at expected
levels but theTatB ⁄ 3Gly mutations result in a much-
reduced TatB signal, presumably reflecting problems in
insertion and ⁄ or stability. No TatC signal is evident,
but this again appears to reflect problems in detection
of the Strep-tag II as this mutant is active in Tat-
dependent transport (see below).
C. M. L. Barrett and C. Robinson Mutagenesisof TatABC
FEBS Journal 272 (2005) 2261–2275 ª 2005 FEBS 2267
Figure 6 shows TorA and TorA-GFP export assays
for these TM span mutants. OftheTatA mutants,
TatA ⁄ I12P shows no detectable defect because GFP
and TorA are found predominantly in the periplasm.
The TatA ⁄ 3Gly mutant, on the other hand is blocked
in export and no translocation activity can be detected.
The same applies to theTatA ⁄ 3Pro6Gly mutant,
although this is expected because the immunoblots in
Fig. 5 show no signal for TatA. The real surprise is
the TatA ⁄ 3Pro3Gly mutant, which is shown to export
both TorA and TorA-GFP with high efficiency. Given
that the parent TatA ⁄ 3Gly mutant is completely inac-
tive, this result indicates that the three proline residues
somehow compensate forthe inhibitory effects of the
glycine residues and enable translocation to occur.
Finally, Fig. 6 shows that the two TatB mutants,
TatB ⁄ 3Gly andTatB ⁄ V12P, are highly active in
export; perhaps surprisingly given the drastic effects of
the 3Gly mutations in TatA.
We also tested the effects of expressing tatABC
operons carrying combinations of these mutations in
the TM spans of both TatAand TatB, namely (TatA ⁄
3Gly + TatB ⁄ 3Gly) (TatA ⁄ 3Gly + TatB ⁄ V12P) (TatA ⁄
I12P + TatB ⁄ 3Gly) and (TatA ⁄ I12P + TatB ⁄ V12P).
Immunoblots confirmed that theTatABC were syn-
thesized at approximately the same levels as the wild-
type subunits generated from pBAD-ABC (data not
shown), and activity assays are shown in Fig. 7. With
the TorA assays, the control tests show efficient export
with wild-type TatABCand a complete block in export
in the tat null mutant (denoted DABCDE), as expected.
The combination of (TatA ⁄ 3Gly + TatB ⁄ 3Gly) is
blocked in Tat function, and this is not unexpected
given that theTatA ⁄ 3Gly mutant itself shows no
export activity. However, a combination of (TatA ⁄
3Gly + TatB ⁄ V12P) displays very efficient export of
TorA and this shows that theTatB ⁄ V12P mutation
compensates forthe drastic effects ofthe 3Gly muta-
tions in TatA. This is confirmed by the TorA-GFP
export assays, which reveal a complete block in export
with the (TatA ⁄ 3Gly + TatB ⁄ 3Gly) mutant but near
wild-type export efficiency with (TatA ⁄ 3Gly + TatB ⁄
V12P). The remaining (TatA ⁄ I12P + TatB ⁄ 3Gly) and
(TatA ⁄ I12P + TatB ⁄ V12P) mutants export both TorA
and TorA-GFP efficiently, in keeping with the finding
that none ofthe mutations affect export to a detect-
able extent when present in TatA or TatB alone (see
Fig. 6). In conjunction with the data shown in Fig. 6,
these data show that the severe effects of the
TatA ⁄ 3Gly mutations can be rescued by the presence
of additional mutations either elsewhere in the TatA
TM span or in theTatB TM span (TatB ⁄ V12P
mutation).
Mutagenesis of TatC
TatC has also been studied in previous reports and a
number of mutations have been characterized [26,27],
but some apparent differences were reported in studies
on the same mutants by different groups (see below).
Few TatC residues are highly conserved but of these, a
high proportion is clustered in the N-terminal cyto-
plasmic domain andthe first cytoplasmic loop. In our
previous analysis [26] only two residues were found to
A
B
Fig. 5. Expression ofTatAandTatB mutants containing alterations
in the transmembrane spans. (A) Sequences ofthe TM regions of
TatA and TatB, with residues numbered andthe substitutions indi-
cated. (B) Expression ofthe various mutations constructed within
the pBAD-ABC vector. Samples were analyzed by immunoblotting
with antibodies to TatA, TatBandthe Strep-tag II on TatC. Mobili-
ties of molecular mass markers are given on the left.
Mutagenesis ofTatABC C. M. L. Barrett and C. Robinson
2268 FEBS Journal 272 (2005) 2261–2275 ª 2005 FEBS
be absolutely essential for TatC function: R17A
(N-terminal cytoplasmic region) and P48A (first peri-
plasmic loop). The deletion of three residues (20–22)
also disrupted function, prompting the suggestion that
this cytoplasmic domain played an important role. In
a separate study [27], R17 was again found to be
important but two acidic residues, E103 and D211
were found to be particularly critical; E103A ⁄ Q ⁄ R
mutants and D211A were completely inactive,
although D211E ⁄ N mutants were partially active.
These residues are located on the first cytoplasmic loop
and third periplasmic loop, respectively. Substitution
of F94 (at the interface between cytoplasmic loop I
and the membrane bilayer) was also reported to block
transport activity [27]. D211 was not analyzed in our
previous study [26] but we did find that E103A showed
no translocation defect at all, and so we have made
new mutants in all three residues in order to analyse
the effects using our expression and assay systems.
As with other mutants, we carried out expression
studies using all ofthe TatC mutants; in each case the
TatABC subunits were present at similar levels and in
A
B
Fig. 6. Effects of mutations in the TM
regions ofTatAand TatB. TheTatA and
TatB mutants containing alterations in TM
spans (detailed in Fig. 5) were tested for
effects on translocation activity. (A) Muta-
tions within the pBAD-ABC vector were
assayed for export of TorA. (B) mutations
within pEXT-ABC were assayed for export
of TorA-GFP, as detailed in Fig. 2. Cells
expressing pBAD-ABC or pEXT-ABC without
mutations were analyzed as controls, and
DtatABCDE cells were analyzed for export
of TorA, again as a control. Other symbols
are as in Fig. 2.
A
B
Fig. 7. A mutation in theTatB TM span can compensate for the
severe effects oftheTatA ⁄ 3Gly mutation. This figure illustrates the
translocation activities of four DtatABCDE strains expressing pBAD-
ABC or pEXT-ABC in which mutations are present in the TM spans
of both TatAandTatB (mutations in TatA are shown underlined and
labels on the right indicate whether the mutations are in TatA or
TatB). Cells were analyzed forthe export of TorA (A) or TorA-GFP
(B) using protocols described in Fig. 2.
C. M. L. Barrett and C. Robinson Mutagenesisof TatABC
FEBS Journal 272 (2005) 2261–2275 ª 2005 FEBS 2269
these instances the TatC Strep-tag II were all found to
be intact (data not shown).
Mutations in the first cytoplasmic loop are charac-
terized in Fig. 8(A). None of these mutants display
any detectable defects in translocation efficiency, inclu-
ding the E103A and E103Q mutants which do not
even contain elevated levels ofthe precursor protein
TorA-GFP. These data do not agree with reports that
the two residues are critical [27], and with this appar-
ent contradiction in mind we made each mutant again
and again found them to be highly active (data not
shown). Figure 8(B) shows the effects of mutations in
TM spans two to six. Most of these mutations again
have no detectable effect with the notable exception of
F94A which is completely inactive. These results agree
with those reported in [27].
The final study on TatC is shown in Fig. 9, where
mutations in the periplasmic loops are analyzed. We
have previously shown that the P48A mutation des-
troys activity, andthe data confirm this result with no
export detected using either assay system. The K73A
and Y154S mutants are active, as expected from previ-
ous studies [26] and so too are the D211A and D211N
mutants that were described as completely or partially
inactive, respectively, in a previous study [27]. In
Fig. 9, no translocation defects are apparent with
either D211A or D211N and these mutants were again
A
B
Fig. 8. Mutations in several residues of the
cytoplasmic loops in TatC cause no detect-
able loss of translocation activity. (A) TatC
mutants carrying substitutions in the 1st
cytoplasmic loops were tested for export of
TorA-GFP as described in Fig. 2; mutations
were made within the pEXT-ABC vector and
the Figure shows assays using pEXT-ABC
as a control. (B) Similar tests carried out
using TatC mutants carrying substitutions in
TM spans (numbered above the lanes).
A
B
Fig. 9. Effects of mutations in the periplasmic loops of TatC. Muta-
tions in the three periplasmic loops of TatC (numbers indicated
above the lanes) were assessed using export assays for TorA
(upper panel) or TorA-GFP (lower panel) as described earlier for
other mutants (Fig. 2).
Mutagenesis ofTatABC C. M. L. Barrett and C. Robinson
2270 FEBS Journal 272 (2005) 2261–2275 ª 2005 FEBS
[...]... Mutations in the TM spans ofTatAandTatB Most ofthe previous studies on TatA ⁄ B have focused on the more highly conserved residues and we considered it important to test the effects of introducing structural rearrangements ofthe TM spans of these subunits It has not been established whether these TM spans simply anchor the amphipathic regions and cytosolic 2272 domains to the membrane, or whether they... translocation activity and this reinforces the importance of this region Truncation analysis has similarly pointed to essential roles for this region in both TatAandTatB [29] While the precise effects ofthe individual sets of mutations await further analysis, the data obtained with new combinations ofTatAandTatB mutants have unexpected and interesting properties For example, theTatA K24A mutant exhibits... with each other, and that these mutations can be safely accommodated in either subunit separately, whereas the simultaneous presence of both mutations leads to an undue disturbance ofthe inter-subunit interaction 2271 MutagenesisofTatABC C M L Barrett and C Robinson Table 1 Primers used for site-specific mutagenesisofTatABC Only the forward primers are shown Name of primer TatA mutants G2A 3Pro3Gly... essential The only mutant devoid of translocation activity in the present study and [25] is TatA ⁄ F39A Mutations in the predicated amphipathic region can have more drastic effects, and we previously analyzed the effects of substituting groups of basic residues in these regions ofTatAandTatB [24] The present study has increased our understanding of this region in two ways First, the removal of three... levels of TorA and TorA-GFP export, as does theTatB 2R > N mutant However, a combination ofthe two mutations leads to a near-complete loss of TorA export and an absolute loss of TorA-GFP export The same phenomenon is observed with the combination of K24A in TatAand 3K > Q in TatB, each of which exhibits no translocation defects in isolation We propose that these regions interact with each other, and. .. in participating in the translocation channel, although further studies are certainly required to obtain a clear picture ofTatB function Again, an analysis of mixed TatA ⁄ TatB mutations provides strong indications for critical interactionsbetween these subunitsTheTatA 3Gly mutant is unable to export either TorA or TorA-GFP, but the simultaneous presence ofthe V12P mutation in TatB restores export... results described above, these data provide indirect evidenceofinteractionsbetween at least two regions ofTatAand TatB: the predicted amphipathic regions andthe TM spans It will be important to probe these issues in detail because little is currently known about the molecular details ofthe proposed TatATatB interaction under conditions where TatABC are expressed at physiological stoichiometries... h growth the cells were analyzed by fractionation as detailed below For studies on the export of TorA-GFP, cells expressing pJDT1 and pEXTABC were grown for 2 h in the presence of 50 lm arabinose to induce expression of TorA-GFP, andthe cells were then pelleted and resuspended in fresh medium containing 1 mm isopropyl thio-b-d-galactoside This leads to induction ofTatABC synthesis fromthe pEXT-ABC... studies [17,37] have shown that TatAandTatB are present within a single complex but these studies did not prove that TatAandTatB actually contact each other A more direct crosslinking approach found evidenceTatA dimers and trimers, as well as TatB dimers, but no TatA- TatB cross-links were observed [31] FEBS Journal 272 (2005) 2261–2275 ª 2005 FEBS MutagenesisofTatABC TatC mutants Most TatC mutations... methods, because in [27] the mutated subunits were generated in a background of wild-type levels ofthe remaining Tat subunits These residues are of real interest because acidic side-chains could participate in proton translocation or, in the case of E103, in the binding ofthetwin-arginine motif in the signal peptide The absence of any translocation defects leads us to conclude that these residues are . Evidence for interactions between domains of TatA
and TatB from mutagenesis of the TatABC subunits
of the twin-arginine translocase
Claire. in
the TM spans of both TatA and TatB, namely (TatA ⁄
3Gly + TatB ⁄ 3Gly) (TatA ⁄ 3Gly + TatB ⁄ V12P) (TatA ⁄
I12P + TatB ⁄ 3Gly) and (TatA ⁄ I12P + TatB