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1
Structural features of the TatC membrane protein that determine docking
and insertion of a twin-arginine signal peptide
Anne-Sophie Blümmel1,2,3, Friedel Drepper4,5, Bettina Knapp4, Ekaterina Eimer1,3,
Bettina Warscheid4,5, Matthias Müller1,6*, and Julia Fröbel1,6
1Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of
Freiburg, 79104 Freiburg, Germany
2Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 79104
Freiburg, Germany
3Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
4Institute of Biology II, Biochemistry – Functional Proteomics, Faculty of Biology, University
of Freiburg, 79104 Freiburg, Germany
5BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg,
Germany
6 Both authors contributed equally
Running title: Functional carboxyl residues of TatC
*To whom correspondence should be addressed. Tel.: 49-761-2035265; Fax: 49-761-
2035274; E-mail: matthias.mueller@biochemie.uni-freiburg.de
Key words: Dicyclohexylcarbodiimide (DCCD, DCC); Escherichia coli; Mass spectrometry;
Membrane protein; Protein cross-linking; Protein export; Protein targeting; TatC; Twin-
arginine translocation
Twin-arginine translocation (Tat)
systems transport folded proteins across
cellular membranes with the concerted
action of mostly three membrane
proteins: TatA, TatB, and TatC. Hetero-
oligomers of TatB and TatC form
circular substrate-receptor complexes
with a central binding cavity for twin
arginine-containing signal peptides.
After binding of the substrate, energy
from an electro-chemical proton
gradient is transduced into the
recruitment of TatA oligomers and into
the actual translocation event. We
previously reported, that Tat-dependent
protein translocation into membrane
vesicles of Escherichia coli is blocked by
the compound N,N’-
dicyclohexylcarbodiimide (DCCD,
DCC). We have now identified a highly
conserved glutamate residue in the
transmembrane region of E. coli TatC,
which when modified by DCCD
interferes with the deep insertion of a
http://www.jbc.org/cgi/doi/10.1074/jbc.M117.812560The latest version is at
JBC Papers in Press. Published on October 31, 2017 as Manuscript M117.812560
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
2
Tat signal peptide into the TatBC
receptor complex. Our findings are
consistent with a hydrophobic binding
cavity formed by TatB and TatC inside
the lipid bilayer. Moreover, we found
that DCCD mediates discrete
intramolecular cross-links of E. coli
TatC involving both its N- and C-tails.
These results confirm the close
proximity of two distant sequence
sections of TatC proposed to concertedly
function as the primary docking site for
twin-arginine signal peptides.
The twin-arginine translocation
(Tat*) system has the remarkable ability to
transport folded proteins across cellular
membranes. It is found in the cytoplasmic
membranes of bacteria and archaea and the
thylakoid membrane of chloroplasts. Tat-
substrates are characterized by the highly
conserved consensus motif SRRxFLK in
their signal peptides (reviewed in (1-6)).
In E. coli, the Tat-translocon
consists of the single spanning membrane
proteins TatA, TatB and TatE and the
hexahelical TatC. TatA and TatB share a
similar core structure. A transmembrane
helix (TM), too short to span the bilayer
entirely, is linked through a short hinge
region to an amphipathic helix that is
followed by a C-terminal domain of
different size (7-10). The six helices of
TatC are tilted within the membrane and
most of them are kinked forming the
concave structure of a cupped hand
(11,12). It is not clear whether the cavity
thus formed is filled with lipids or water.
A TatABCE-complex was shown
through fluorescence microscopy of living
E. coli cells to assemble on demand (13-
15). TatB and TatC interact in a 1:1
stoichiometry (16) and several of these
TatBC protomers form a receptor complex
for a Tat-precursor (17,18). Through TatB
intercalating between two neighboring
TatC monomers, circular TatBC receptor
complexes are formed (19,20), in which
TatB was proposed to form the inner and
TatC the outer shell of a dome-like
structure. A current model of the TatBC
complex is depicted in Fig. 1 (looking at its
trans-sided surface; a side view of TatC
and TatB molecules and their relative
positions within the lipid bilayer is shown
in Fig. 3a). Neighboring TatC monomers
interact through the TM of TatB as well as
via their periplasmic loops (19,21-23).
TatA is found at the periphery of the
complex (19,22,24).
Both TatB and TatC recognize a
Tat-signal peptide in a concerted fashion
(19,25-29). The RR-motive is first
recognized by the N-terminal domain and
the TM 2/TM 3 loop of TatC
(21,25,26,30). Subsequently, a Tat-signal
peptide inserts deeply into a TatB/TatC-
walled cavity (19,24,27-29,31,32), the
conformation of which in turn is
influenced by the signal peptide itself (29).
Upon substrate binding, TatA is
thought to promote the actual translocation
step by either forming a translocation pore
(reviewed in (3)) or destabilizing the
membrane (8,33,34). Both, recruitment of
TatA oligomers as well as the thereby
triggered translocation event require the
proton-motive force (PMF) as sole energy
source.
N,N’-Dicyclohexylcarbodiimide
(DCCD, DCC) was previously shown to
act as an inhibitor of the E. coli Tat-system
by preventing the binding of a Tat-
substrate to the Tat-translocase (35). In
screening E. coli TatC for potential binding
sites of DCCD, we now discovered that
modification by DCCD of the highly
conserved and deeply membrane-
embedded glutamyl residue 170 interferes
with the insertion of a Tat-signal peptide
into the TatBC-complex. In addition,
DCCD-mediated intramolecular cross-
linking of TatC revealed conformational
details of the RR recognition site of E. coli
TatC.
Results
Glutamate 170 of E. coli TatC becomes
quantitatively modified by DCCD
3
DCCD is known to modify
carboxyl side chains that are located in
hydrophobic regions of proteins giving rise
to N-acyl urea adducts (36,37)
(Supplemental Fig. S1a). In order to
identify potential DCCD-reactive carboxyl
side chains of TatC, E. coli membrane
vesicles containing overexpressed TatA,
TatB and a His-tagged TatC variant were
treated with DCCD in the absence of
substrate, and TatC was subsequently
purified by affinity chromatography and
SDS-PAGE. Peptides derived from a
combined digestion of monomeric TatC
with trypsin and chymotrypsin were
analyzed by liquid chromatography-
tandem mass spectrometry (LC-MS/MS).
Data analysis consisted of comparison of
the data with known protein sequences and
chromatographic peak integration using the
MaxQuant program taking into account
possible modifications by DCCD. The
recovery of TatC peptides and their
cumulative MS intensities are plotted in
Fig. 2a along the E. coli TatC sequence,
and the theoretical as well as the
experimentally verified trypsin and
chymotrypsin cleavage sites of TatC are
depicted in Supplemental Fig. S2.
Sequence coverage of TatC was 90.3%
with the three gaps indicated in Fig. 1a and
Supplemental Fig. S2. The first one
flanked by K18 and F37 represents the
hydrophobic stretch of TM1a and the third
between K191 and V196 is located at the
beginning of TM5 (Supplemental Fig. S2).
Whereas these two sections of TatC were
also missing in the MS/MS spectra
obtained from an untreated TatC sample
(not shown), the central gap (K101-R105)
was due to the treatment with DCCD as
demonstrated below. Except for E103,
which is further discussed below, the non-
recovered sequence sections of TatC were
devoid of Asp and Glu residues as
potential target sites for DCCD.
The vertical bars in Fig. 2a mark
all amino acid residues of E. coli TatC that
were found to carry the additional mass of
dicyclohexylurea (DCU, cf. Supplemental
Fig. S1a), namely E4, D63, E170, E244
and D248 (also highlighted in the structure
representation of E. coli TatC shown in
Fig. 2b) and the lengths of these bars
indicate the cumulative MS intensities of
peptides harboring the DCU modification
at the respective positions. Relative to the
intensities of the non-modified peptides,
the lengths of the bars reflect the extent by
which modification through DCCD
occurred. Virtually 100% of the peptide
E170VPVAIVLL178 (Fig. 2a) contained the
DCCD-derived modification, whereas
modification of E244 was observed with
less than 50% of total intensity of the
respective peptide and those of the others
with 10% or lower. The validity of the
modification of E170 of TatC by DCCD is
further documented in Supplemental Fig.
S3 depicting properties of the isolated
peptide E170VPVAIVLL and its MS/MS-
generated fragments containing or lacking
the DCU moiety. Collectively these data
demonstrate that the TM 4 residue E170 of
TatC that is positioned in the middle of the
lipid bilayer (Fig. 2b), represents the
predominant DCCD target of E. coli TatC.
Labeling of TatCE170 with DCCD
could further be demonstrated using the
fluorescent analogue of DCCD, NCD-4
(N-cyclohexyl-N’-(4-dimethylamino-
alpha-naphthyl) carbodiimide)
(Supplemental Fig. S1b) (38). For this
purpose, inside-out inner membrane
vesicles (INV) of E. coli containing
TatABC at overexpressed levels were
treated with NCD-4, either directly or after
pre-incubation with a 10-fold molar excess
of DCCD. Membrane proteins were then
separated by SDS-PAGE and inspected
under UV-light. Two bands became
fluorescently labeled with NCD-4, unless
DCCD was also present, indicating
DCCD-specific binding sites in both
proteins (Fig. 2c, lanes 1, 2). The lower of
the two bands was of the size of TatC.
Accordingly, its labeling with NCD-4 was
drastically reduced when membrane
vesicles were used that had E170, the
major DCCD target of TatC, exchanged
against alanine (lane 3). As demonstrated
4
in Fig. 2d, this decrease in labeling with
NCD-4 could not be accounted for by
reduced TatC levels in the TatCE170A
vesicles but must have been caused by the
E170A mutation of TatC. These results
therefore prove the identity of the lower
band with TatC and confirm the
accessibility of TatC residue E170 to
DCCD and NCD-4. The residual labeling
by NCD-4 of TatC carrying the E170A
mutation (Fig. 2c, lane 3) is most likely
due to binding of NCD-4 to one or more of
the minor DCCD targets revealed by mass
spectrometry. The upper band that became
labeled by NCD-4 was of the size of TatB
and in fact was not obtained for TatB-
lacking membrane vesicles (lane 5). Thus
obviously also TatB contains DCCD-
sensitive residues but this is not subject of
this study.
DCCD interferes with the proper
accommodation of a Tat signal peptide
within the TatBC binding cavity
Upon binding, the RR-consensus
motif of a Tat signal peptide is recognized
by surface-exposed residues of the N-tail
and the TM 2/TM 3 loop of TatC
(12,21,22), whilst the downstream part of
the signal peptide inserts as a hairpin into a
TatBC-formed cavity (19,24). In this
cavity, it contacts the N-terminus of TatB
(19,27,28) as well as trans-sided residues
of the TM 5 of TatC (19,24). In accordance
with a current model (1), Fig. 3a illustrates
how an RR-precursor might be
accommodated in the TatBC receptor
complex. In order to understand, how
DCCD might affect this binding step, we
synthesized and radioactively labeled the
model Tat substrate TorA-mCherry (39) in
vitro in the presence of TatABC-containing
INV carrying the photo-activatable cross-
linker p-benzoyl-phenylalanine (Bpa)
either in the non-helical N-tail of TatB or
at the internal binding site of TatC (Fig.
3a, residues marked in red). As shown in
Fig. 3b, Bpa variants at positions F2, I4,
and F6 of TatB when exposed to UV-light
yielded a prominent radioactive 60 kDa
product representing the adduct of one
TatB molecule to the radioactively labeled
37 kDa precursor TorA-mCherry (green
star, TatB x TmC). In addition, higher
molecular mass adducts were obtained
representing adducts between precursor
and more than one TatB molecule (green
stars). This follows from the fact that they
carry the radioactive label of the precursor
and must contain a Bpa-hosting protein,
the only one of which is TatB in this setup
(19). These adducts did not form, or were
at least drastically diminished, in the
presence of DCCD (Fig. 3b, lanes 3, 6, 9).
Similarly, when Bpa was replacing the
trans-sided residues V202, L206, and T208
in the TM 5 of TatC, 1:1 cross-links
between TorA-mCherry and TatC were
obtained (Fig. 3c, blue star). Again DCCD
considerably interfered with the formation
of these adducts (lanes 3, 6, 12). This was,
however, not the case when Bpa had been
incorporated into the superficial binding
site for the consensus motif of Tat signal
peptides at position L9 of TatC (lanes 14
and 15).
This latter result demonstrates that
DCCD does not prevent binding of a Tat
substrate to TatC but specifically seems to
impair its hairpin-like insertion into the
TatBC binding cavity. Notably, when
glutamate 170, the main target site of
DCCD in TatC, had been mutated to
alanine, DCCD hardly interfered with
precursor binding to TatC206 (Fig. 3c,
compare lanes 5, 6 with 8, 9). This result
indicates that the major reason for DCCD
blocking precursor insertion was
modification of E170 of TatC. Different
from DCCD, the protonophore CCCP
(carbonyl cyanide m-chlorophenyl-
hydrazone) did not prevent cross-linking
between TorA-mCherry and residue 206 of
TatC (Fig. 3c, compare lanes 17 and 19),
although it efficiently blocked transport of
TorA-mCherry into INV as indicated by
the lack of signal sequence processing (m-
form of TorA-mCherry, lanes 18, 19). This
rules out that DCCD inhibited the binding
of TorA-mCherry to the internal binding
site of TatC via dissipation of the proton-
5
motive force (PMF), which DCCD causes
by default through blockage of the vesicle-
bound F1FO-ATPase (40).
In order to further demonstrate that
DCCD, by binding to E170 of TatC,
interfered with the hairpin-like insertion of
a Tat signal peptide into the TatBC binding
cavity, we analyzed the interaction
between Tat precursor and TatBC also by
incorporating the cross-linker Bpa into the
TorA signal sequence at the two sites
highlighted in Fig. 4a. As shown in
numerous previous reports
(19,21,25,26,35,41), the consensus motif,
represented in Fig. 4a by the F14Bpa
mutation of TorA-mCherry, cross-links to
TatC (Fig. 4b, lane 2, blue star). On the
contrary, the hydrophobic core of the
signal peptide, represented in Fig. 4a by
the L27Bpa mutation, cross-links to TatB
and to some degree also to TatA (Fig. 4b,
lane 8, green and pink stars). DCCD did
not interfere with cross-linking of the
F14Bpa variant of TorA-mCherry to TatC
(Fig. 4b, compare lanes 2 and 3). This is
totally consistent with the finding shown in
Fig. 3b that binding of TorA-mCherry to
L9 located within the RR-recognition site
of TatC was unaffected by DCCD.
In contrast, cross-linking of the
L27Bpa variant of TorA-mCherry to TatB,
as well as to TatA, was strongly reduced
when DCCD was added (Fig. 4b, lanes 8,
9). Instead, an adduct of the size of TorA-
mCherry cross-linked to TatC appeared
(lane 9, blue star), similar to what we
previously observed for INV that totally
lacked TatB (19). This DCCD-caused
reversal of contacts between the signal
peptide and TatB and TatC was largely
abolished when membrane vesicles were
used that contained the E170A variant of
TatC (Fig. 4b, green and blue stars,
compare lanes 9 and 12). These findings
confirm that DCCD interferes with the
proper insertion of a Tat substrate into the
TatBC binding cavity through modification
of residue E170 of TatC. Again, DCCD did
not cause these disturbances by dissipating
the PMF. This follows from the data
shown in Fig. 3c that in contrast to DCCD,
the protonophore CCCP did not diminish
cross-linking of the RR-precursor to TatC
(blue star) and TatB (green star), although
it abolished the PMF-sensitive interaction
of the L27Bpa variant of TorA-mCherry
with TatA (pink star).
Interference of DCCD with the
proper insertion of a Tat signal peptide into
the TatBC binding cavity could further be
demonstrated using a different strategy. In
Fig. 5, TorA-mCherry was synthesized and
radioactively labeled in vitro. In the
presence of TatABC vesicles (Fig. 5a, lane
1), about half of the precursor of TorA-
mCherry (p) was found processed to the
mature form (m). Because of its resistance
towards proteinase K (lane 2) the m-form
must have been translocated into the lumen
of the vesicles. By the same criterion, also
some non-processed precursor was found
translocated, although this fraction of
cursor was partially digested by proteinase
K removing a few amino acids from the N-
terminus of the membrane-embedded TorA
signal peptide (22). Dissipation of the PMF
by CCCP (lane 4) and impairment of
signal peptide insertion by DCCD (lane 6)
totally prevented the accumulation of any
proteinase K-resistant p- and m-forms of
TorA-mCherry. Similarly, when instead of
TatABC vesicles, TatAC vesicles were
used, translocation of TorA-mCherry was
also completely abolished, now due to the
missing TatB (lane 8).
Nevertheless TatB-deficient
vesicles allowed for the appearance of the
m-form of TorA-mCherry (lane 7). As
previously reported (31), in the absence of
TatB, TatC obviously inserts the TorA
signal peptide across the membrane so that
it becomes prematurely cleaved off by
signal peptidase without prior translocation
of the Tat substrate into the vesicles. Upon
treatment of TatAC vesicles with DCCD,
the premature cleavage of the TorA signal
peptide dropped from 41% to 16%
(compare lanes 7 and 11). Again, this
inhibitory effect of DCCD was not based
on DCCD dissipating the PMF of the
vesicles, because the uncoupler CCCP did
not affect processing of TorA-mCherry by
6
TatAC vesicles to any significant degree
(lane 9). However, in TatAC vesicles
carrying the TatCE170A variant, DCCD did
not any longer interfere with the premature
processing of TorA-mCherry (Fig. 5b,
compare lanes 1 and 6) indicating that also
in the absence of TatB, DCCD impairs the
insertion of a Tat signal peptide into the
membrane via modifying the E170 residue
of TatC.
Intramolecular cross-linking by DCCD
reveals conformational details of the RR
recognition site of E. coli TatC
As mentioned above for our LC-
MS/MS analysis of TatC, the linear peptide
sequence H102E103R104 from the largely
hydrophilic TM 2/TM 3 loop of TatC was
not recovered when TatC had been treated
with DCCD (Fig. 2a, Supplemental Fig.
S2). In theory, this could be explained if
DCCD caused an intramolecular cross-link
between the missing peptide and another
part of the same TatC molecule thereby
generating a branched peptide. As
illustrated in Supplemental Fig. S1a,
adducts of DCCD to free carboxyl side
chains form via a reactive intermediate. If
this intermediate is attacked by a nearby
primary amine, DCCD is released as
dicyclohexylurea and an amide
(isopeptide) bond between the original
carboxyl group and the attacking amino
group is generated. In fact, the LC-MS/MS
analysis of DCCD-treated TatC revealed
two branched peptides involving the
K101HER104 peptide sequence of the TM
2/TM 3 loop of TatC (Fig. 6a). One
originated from a cross-link of E103 to the
α-amino group of the N-terminal
octapeptide S2VEDTQPL9 of TatC,
whereas the other encompassed the C-
terminal tetradecapeptide
N242REEENDAEAESEK255 of TatC cross-
linked via E244 to K101 of the KHER
peptide. Supplemental Fig. S4a,b
demonstrates the identification of both
branched peptides via their MS/MS-
generated fragments. Both products were
not detected in the MS data obtained from
non-treated TatC (Fig. 6a, green curve)
demonstrating that they resulted from the
cross-linking activity of DCCD.
These findings indicate that besides
the five carboxyl side chain residues E4,
D63, E170, E244, D248 of TatC, which
became modified by DCCD (Fig. 2a),
E103 is an additional target for DCCD.
Moreover, the DCCD-mediated
intramolecular cross-linking of TatC
provides evidence for a close proximity of
the cytosolic TM 2/TM 3 loop sequence
K101HE103 to both cytosolically oriented N-
and C-tails of TatC. The conformations of
the N- and C-tails of E. coli TatC, which
are longer than those of the Aquifex
aeolicus TatC, have not been ascertained
thus far. The DCCD-mediated
intramolecular TatC cross-links obtained
now suggest that the N-terminal and C-
terminal domains might fold back on the
core structure of TatC (Fig. 6b) thereby
contributing to the compact fold of the
TatC molecule. Such an orientation is also
supported by the identification of an
isopeptide resulting from a DCCD-caused
cross-link between S2 and E244 of TatC
(Supplemental Fig. S4c). Moreover, the
juxtaposition of the N-tail and the TM
2/TM 3 loop is fully consistent with both
cytosolic domains of TatC constituting the
decoding area of TatC for the RR-pair of
the Tat signal peptide (12,21,22).
Discussion
In trying to unravel how DCCD
blocks binding of a Tat substrate to the Tat
translocase, we screened TatC for carboxyl
residues that became modified by DCCD
and identified glutamate 170 as a major
target of DCCD. DCCD treatment of TatC
was performed in the absence of added
substrate because of the insufficiently tight
interaction of an RR-signal peptide with
purified TatC (12) potentially causing
heterogenous TatC populations.
Modification of E170 by DCCD perturbed
the signal peptide’s interaction with trans-
sided residues of TatB (N-tail) and TatC
(distal part of TM 5). Vice versa, in the
presence of DCCD, the hydrophobic core
7
of the TorA signal peptide (represented by
L27) did not any more reach out to TatB
but rather contacted TatC. Furthermore,
modification of TatCE170 by DCCD
interfered with the insertase function of
TatC, a property that can be experimentally
demonstrated by use of membrane vesicles
lacking TatB. In this artificial situation,
TatC directly transfers RR-precursors to
the trans-sided signal peptidase resulting in
a proteolytic removal of the signal peptide
uncoupled from translocation (31).
Importantly, all these DCCD-caused
alterations were largely reversed by the
TatCE170A mutation indicating that they
directly resulted from DCCD modifying
E170 of TatC.
The DCCD-sensitive residue E170
of TatC is highly conserved among
bacterial TatCs (42). Mutational
replacement of this glutamate residue
impairs Tat-specific transport but does not
eliminate it (22,41,43). A possible role of
TatCE170 in the binding of the SRRxFLK
consensus motif has been discussed (2,11).
By contrast, cross-linking studies have not
disclosed any vicinity of TatCE170 neither
to Tat substrates nor to TatB (22). In
chloroplast TatC (cpTatC), the residue
three positions upstream of the E170
equivalent yielded disulfide bonds with the
TM of chloroplast TatA (Tha4) and this
contact was dependent on the presence of a
Tat substrate and the PMF (24). These
results would be consistent with the idea
that protonation events might allow
TatCE170 to form a hydrogen bond with the
TM of TatA (12).
All studies performed thus far
exclude E170 of TatC as a direct binding
partner of a Tat signal peptide. Therefore
impaired precursor binding to DCCD-
modified TatC is unlikely to be caused by
DCCD masking E170 as a possible
interaction site of the cross-linker Bpa. We
rather assume that the bulky DCU moiety
when attached to TatCE170 sterically blocks
the insertion of an RR-precursor into the
TatBC binding cavity. This is also
suggested by the model depicted in Fig. 3a,
where E170 of the left-hand TatC
monomer would be close to the signal
peptide shown, provided that our hand-
crafted position of the signal peptide
comes near to the actual molecular
situation. In the crystal structure of TatC,
E170 is predicted to form hydrogen bonds
with TM 2 and 3 in the back of the
molecule (12). Obviously, the most
prominent feature of TatCE170 is its
location in the interior of the bilayer,
where according to MD simulations it is
hydrated and thus perturbs the bilayer
structure (11,12). Conversely, modification
of TatCE170 by DCCD as established here
would require that it is accommodated in a
rather hydrophobic environment. Blocking
insertion of a Tat signal peptide deeply into
the membrane by modifying E170 by
DCCD, suggests that the area around this
glutamyl residue is part of, or at least very
close to, the binding pocket for the Tat
signal peptide, which TatC and TatB
concertedly form. That at least discrete
patches of this binding pocket are
hydrophobic in nature is strongly
suggested by recent findings indicating that
the hydrophobic core of an RR-signal
peptide significantly contributes to a
productive interaction with the TatBC
receptor complex (44).
While blocking insertion of the
signal peptide through interaction with
E170, DCCD did not abolish every contact
of the RR-signal sequence with the TatBC
receptor complex. Cross-linking to position
L9 in the N-terminal domain of TatC was
not disturbed by DCCD, nor was cross-
linking of the consensus motif of the TorA
signal peptide via F14Bpa to TatC. Thus,
in contrast to the insertion of an RR-
precursor into the TatBC binding cavity,
DCCD did not negatively affect docking of
an RR-signal peptide to the Tat
translocase. Remarkably, this was the case,
although DCCD formed an intramolecular
cross-link between the N-tail and the TM
2/TM 3 loop of TatC. These two domains
had previously been identified as
interaction sites for RR-signal sequences
through cross-linking studies as well as the
mapping of E. coli tatC mutations, which
8
suppress inactivating alterations in the RR-
motif. These studies had identified a
number of residues in the N-terminus
(including L9 and going up to Q22) and
the TM 2/TM 3 loop of TatC as being
directly or indirectly involved in
interacting with RR-signal sequences
(21,22,27-30). The exact residues within
these two domains of TatC that directly
interact with the twin-arginines of Tat
signal peptides have not yet been
established (11,12). The composite nature
of the superficial RR-recognition site
involving the non-contiguous N-terminus
and TM 2/TM 3 loop of TatC, is, however,
reinforced by our finding that the covalent
fixation of both domains through DCCD
does not negatively affect docking of an
RR-precursor.
About 50% of all TatC molecules
that were digested with trypsin and
chymotrypsin showed a modification of
E244 by dicyclohexylurea (cf. Fig. 2a).
E244 is located in the flexible C-tail of E.
coli TatC. The finding that E244 becomes
also cross-linked to K101 through DCCD
suggests that this area of the C-terminal
domain of E. coli TatC can move in close
proximity to the TM 2/TM 3 loop.
Moreover, the fact that DCCD attacks
E244 to a significant extent indicates that
this C-terminal stretch of E. coli TatC is
located in a hydrophobic environment of
the TatC molecule, which would be
consistent with its vicinity to the
membrane-enclosed (11,12) TM 2/TM 3
loop. Such a conclusion is also supported
by the DCCD modification of the nearby
D248 residue, although this occurred to a
considerably lower degree than that of
E244 (cf. Fig. 2a). Collectively, these
findings suggest that both, the N- and C-
terminal ends of the E. coli TatC molecule
are in close contact to the helical core of
the molecule and that this conformation is
compatible with its function as a substrate
receptor.
Experimental Procedures
Plasmids
Plasmids used in this study are
listed in Supplemental Table S1. Plasmids
expressing Bpa variants of TatB and TatC
have been described (19). Plasmid p8737
was used to introduce the Ala codon GCG
into tatC and to add a 6His-Tag at the C-
terminus of TatC (p8737-TatABCHis)
using the primers listed in Supplemental
Table S2. Plasmids were amplified using
Pfu Ultra II Fusion HS DNA Polymerase
(Agilent Technologies) according to the
manufacturer’s protocol. Amber stop
codon mutations in the gene encoding
TorA-mCherry of plasmid pPJ3 have been
described (19). T4 DNA Ligase was
purchased from Thermo Scientific. Gel
extraction and DNA extraction kit
(Qiagen) were used for DNA purification.
In vitro reactions
The RR-precursor protein TorA-
mCherry was synthesized and
radioactively labelled by in vitro
transcription/translation using plasmid
pPJ3. Cell extracts used for the in vitro
synthesis were prepared (45) from E. coli
strain SL119 (46) or alternatively from
Top10 (Invitrogen) transformed with
plasmid pSup-BpaRS-6TRN(D286R) to
express amber stop codon mutants of
TorA-mCherry (32). Coupled
transcription/translation reactions were
performed in 50 µL aliquots as described
(45). INV were added 10-15 min after
starting the synthesis reaction and
incubated for 20 min at 37°C.
Assaying protein translocation into
INV by proteinase K protection, addition
of CCCP, and Bpa-dependent cross-linking
by irradiating samples with UV-light for
20 min on ice have been described (39).
DCCD was added to a final concentration
of 0.5 mM before adding INV. SDS-PAGE
using 10% gels was performed as
described (45).
Membrane vesicles
9
Inside-out inner membrane vesicles
(INV) were prepared as described (45)
from E. coli strains BL21(DE3)*
(Novagen) or BL21(DE3)ΔTat (kindly
provided by B. Ize and T. Palmer)
transformed with plasmid p8737 and
derivatives thereof. TatABC-INV
containing Bpa variants of TatA, TatB and
TatC were prepared as described (19).
Purification of DCCD modified TatC
For mass spectrometry analysis of
the DCCD-modified TatC, INV were
prepared from E. coli strains BL21(DE3)*
(Novagen) transformed with p8737-
TatABCHis as described (45) except that
vesicles were finally resuspended in buffer
A (50 mM Tris-HCl pH 7.5, 150 mM
NaCl, 5 % glycerol) and diluted to a
concentration of approximately 10 mg
protein/mL. DCCD was added to a final
concentration of 0.5 mM and incubation
was performed over night at 4°C.
Membrane proteins were solubilized for 1
h at 4°C by NLS (N-Lauroylsarcosine
sodium salt, final concentration 0.33%) in
the presence of 30 mM imidazol. Insoluble
material was removed by centrifugation
(30 min, 36 000 x g, 4 °C). Affinity
purification of TatC was performed using
an Äkta Prime System (Amersham
Bioscience). The solubilized membrane
proteins were loaded on a 5 mL HP His-
trap column (GE-Healthcare) equilibrated
with buffer B (buffer A containing 0.17 %
NLS). Non-specifically bound material
was removed by three washing steps each,
using 30 mM and subsequently 50 mM
imidazol in buffer B. Elution was
performed by applying an imidazole
gradient from 50 – 500 mM imidazole in
buffer B. The eluate was concentrated
using Amicon centrifugal tubes (30 kDa
cutoff, Millipore) and separated by 10%
SDS-PAGE.
In-gel digestion of membrane proteins
Protein-containing bands were
excised from SDS-polyacrylamide gels,
destained, and subjected to reduction of
cysteine residues with 5 mM Tris(2-
carboxy-ethyl)phosphine dissolved in
10 mM NH4HCO3 (incubation for 30 min
at 37°C) and subsequent alkylation of free
thiol groups with 50 mM iodoacetamide in
10 mM NH4HCO3 (30 min at room
temperature in the dark). Monomeric TatC
was in-gel digested at 37 °C overnight
using trypsin and chymotrypsin in 100 mM
Tris-HCl, 10 mM CaCl2, pH 8, by adding
0.25 µg of each protease at the start and
after 4 h of incubation.
Liquid chromatography-tandem mass
spectrometry (LC-MS/MS)
Peptide mixtures were analyzed in
two biological replicates by UHPLC-
MS/MS using an UltiMate 3000
RSLCnano coupled to a Q Exactive Plus
(Thermo Fisher Scientific) mass
spectrometer essentially as described (47),
except for using a 45 min linear gradient
for separation by C18 reversed-phase nano
LC. Peptides were identified by database
searches using the MaxQuant program
(version 1.5.5.1, (48)) and protein
sequences for E. coli TatA, TatB and TatC
as well as for a set of common
contaminants. A maximum of four missed
sites for proteolytic cleavage by trypsin or
chymotrypsin was allowed. Modification
of aspartate or glutamate by
dicyclohexylurea (DCU, +206.17830 Da,
modification-specific neutral loss of -
125.08406 Da) and oxidation of
methionine were defined as variable and
carbamidomethylation of cysteine as fixed
modifications. Peptides were identified
with a minimum length of six amino acids,
a false discovery rate of < 1%, and scores
> 17 (p-value below 0.02) or > 40 (p-value
below 0.0001) for unmodified and
modified peptides, respectively. MS
intensities and peptide scores were read out
from the ‘evidence.txt’ table and summed
up per amino acid position. In order to
estimate the proportion of proteins that are
modified by DCU at a given site,
intensities of DCU-modified peptides
10
identified with a localization probability ≥
0.9 were summed up per modified site. For
identification of cross-linked peptides, the
program pLink (49) was used essentially as
described previously (50), however taking
into account zero-length cross-links (-
18.0106 Da) between aspartate or
glutamate and lysine or the N-terminal
amino group as well as monolink
modifications (+206.1783 Da) of aspartate
or glutamate. Relative quantification
comparing DCCD-treated versus untreated
samples for visualization was done by
extracted ion chromatograms integrating
the first three isotope peaks (mass
tolerance 5 ppm) of each precursor ion
using Xcalibur Qual Browser software (v.
2.2, Thermo Fisher Scientific).
Identification of DCCD binding sites
The binding of DCCD was detected
directly by MS analysis (see above) or
indirectly using the fluorescent DCCD-
analogue NCD-4 (Synchem). To this end,
2.5 µL of each INV preparation (~ 100
A280 u/mL) was diluted with 97.5 µL INV-
buffer (45) and treated with either 5 mM
DCCD (diluted from a 0.5 M stock
solution in DMSO) or DMSO for 10 min at
37 °C before adding 0.5 mM NCD-4
(using a 50 mM solution in DMSO that
had been diluted from a 1 M stock solution
prepared in tetrahydrofurane) to each
sample. Samples were incubated at 37 °C
for 30 min. Proteins were precipitated with
5 % TCA, redissolved in 25 µl SDS-
loading buffer and separated by SDS-
PAGE (12 % gel). After electrophoresis,
gels were irradiated with UV-light. NCD-4
emits light at approximately 450 nm if it is
stimulated with UV-light. The emission
was detected using a Fusion FX-7 Spectra
instrument (Vilber) with a F440 emission
filter (Vilber).
Acknowledgements
This study was supported by SFB746 and
the Excellence Initiative (GSC-4, Spemann
Graduate School) of the German Research
Foundation. Research in the Warscheid
group was funded by the Deutsche
Forschungsgemeinschaft (FOR 1905) and
the Excellence Initiative of the German
Federal & State Governments (EXC 294,
BIOSS). We gratefully acknowledge
MuDe Zou for excellent technical
assistance.
Conflict of interest
The authors declare that they have no
conflicts of interest with the contents of
this article.
Author contributions
Acquisition and analysis of data: A.S.B.,
F.D., B.K., J.F., E.E.
Conception and design: A.S.B., F.D.,
B.W., J.F., M.M.
Drafting and revision of article: A.S.B.,
F.D., B.W., J.F., M.M.
Final approval: J.F., M.M.
*Abbreviations used are: Tat, twin-arginine translocation; PMF, proton-motive force; DCCD
(DCC), N,N’-dicyclohexylcarbodiimide; TM, transmembrane helix; LC-MS/MS, liquid
chromatography-tandem mass spectrometry; DCU, dicyclohexylurea; NCD-4, N-cyclohexyl-
N’-(4-dimethylamino-alpha-naphthyl) carbodiimide; INV, inside-out inner membrane
vesicles; CCCP, carbonyl cyanide m-chlorophenyl-hydrazone; PK, proteinase K; p, precursor
of TorA-mCherry; m, mature form of TorA-mCherry; NLS, N-Lauroylsarcosine
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Figure legends
Figure 1. Model of the Tat translocase. The hexahelical x-ray crystallographic structures of
four TatC monomers are shown in different shades of blue with the six transmembrane helices
numbered for the monomer shown in cyan. A tetrameric structure was chosen for reasons of
clarity, whereas experimental data for higher order assemblies have been provided. The
isolated transmembrane helices of four TatB molecules (green) and eight TatA molecules
(red) were modeled against the TatC structure according to experimentally verified contact
sites. The side-by-side arrangement of two neighboring TatC monomers is also derived from
cross-linking studies. View is from the trans-side down the membrane normal with substrates
(not shown) approaching from below.
Figure 2. Glutamate 170 of E. coli TatC becomes quantitatively modified by DCCD. (a)
Sequence coverage and sites of modification by DCCD of E. coli TatC analysed by
quantitative mass spectrometry. For each amino acid along the sequence of TatC, MS
intensities of peptides containing this residue were summed up and plotted against its
sequence residue number. Relative quantification of peptides was performed based on
detected ion intensities. Peptide scores are indicated and reflect the probability-based
significance of peptide identifications based on matched fragment ions in MS/MS spectra.
The sum of peptide scores per amino acid is decoded by the size of the symbols. Indicated are
amino acids flanking the three sequence sections of DCCD-treated TatC that were not covered
by the MS/MS analysis (arrows). Cumulative intensities of peptides identified with
modification of aspartic or glutamic acid by DCU are plotted at positions of the modified sites
(diamonds on vertical lines) according to the same logarithmic scale as the total sum of
intensities including modified and unmodified peptides. Virtually 100% of the peptide
E170VPVAIVLL178 depicted was found modified by DCU. (b) Model of the E. coli TatC
structure adapted from the crystal structure of A. aeolicus TatC (PDB: 4B4A and 4HTT)
(11,12) with the six transmembrane helices and the N- and C-termini marked. Residue E170,
which is virtually completely modified by DCCD, is marked in red, the other DCCD sensitive
residues are marked in blue. The bilayer is outlined by grey bars, the cis-side corresponds to
the cytoplasmic face of E. coli TatC. (c) Vesicles (INV) were prepared from E. coli strains
overexpressing TatABC (wt), TatABCE170A, and TatAC. INV were either treated with DCCD
or DMSO before incubating with the fluorescent DCCD analog NCD-4. Proteins were
separated by SDS-PAGE and analyzed under UV-light. (d) Western blot analysis using anti-
TatC antibodies to demonstrate unimpaired expression of the E170A variant of TatC.
Figure 3. Influence of DCCD on the binding of a Tat-substrate to TatB and TatC. (a)
Model of a signal peptide inserting into the TatBC-complex according to (1). The Tat signal
peptide attaches via its RR-consensus motif to the cis-sided signal peptide binding site of the
left TatC protomer represented by residue L9 and contacts the transmembrane helix (TM) of
TatB and TM 5 of the adjacent TatC molecule. Labeled are residues of TatC and TatB that
interact with the Tat substrate, as shown in (b) and (c) and previously (19,22). The DCCD-
sensitive TatC residue E170A is indicated. (b) Autoradiography. TorA-mCherry (TmC) was
synthesized and radioactively labeled in vitro. Samples were either treated with DMSO or
DCCD and vesicles (INV) harboring the indicated Bpa-variants of TatB were added. For
cross-linking, samples were irradiated with UV-light. Green stars, TatB-TmC adducts. (c) as
in (b) using INV with the indicated Bpa-variants of TatC or in addition with the TatCE170A
substitution. When indicated, samples were treated with 0.1 mM CCCP after synthesis. The
lower panel of lanes 16 – 19 is a lighter representation of the upper autoradiograph with a
better resolution of precursor (p) and mature form (m) of TmC. Blue stars, TatC-TmC
adducts; black star, unidentified adduct (19).
15
Figure 4. Influence of DCCD on signal peptide binding to the TatABC-receptor
complex. (a) Amino acid sequence of the TorA-signal peptide. Amino acids that were
replaced by Bpa are marked in red. (b, c) Autoradiographies. TorA-mCherry (TmC) variants
harboring Bpa at the indicated positions were synthesized and radioactively labeled in vitro.
Samples were treated with DMSO, DCCD and CCCP as indicated prior to incubation with
TatABC (wt) vesicles (INV) or TatABC INV harboring the TatCE170A mutation. For cross-
linking, samples were irradiated with UV-light. p, precursor of TmC; blue, green and pink
stars, adducts of TmC to TatC, TatB and TatA, respectively.
Figure 5. Influence of DCCD on the premature cleavage of a Tat-signal peptide in the
absence of TatB. Autoradiographies. TorA-mCherry was synthesized and radioactively
labeled in vitro. (a) Samples were treated with DMSO, CCCP or DCCD as indicated, before
adding vesicles (INV) containing either TatABC or only TatA and TatC. Upon addition of
TatABC vesicles, the precursor of TorA-mCherry (p) is processed to the mature form (m).
Transport into INV is indicated by proteinase K (PK) resistance. The PK-resistant precursor is
of slightly reduced size because of the removal a few N-terminal amino acids by PK. When
TatAC vesicles were used, digestion of precursor (p) and mature form (m) by PK indicates a
lack of transport due to the missing TatB. The premature processing of the precursor to the
mature form was quantified from three independent experiments. (b) Samples were treated as
in (a) before adding vesicles containing only TatA and the TatCE170A variant.
Figure 6. Intramolecular cross-linking by DCCD reveals conformational details of the
RR recognition site of TatC. (a) Extracted ion chromatograms of cross-linked TatC peptides
analyzed by LC-MS. E. coli TatC treated with DCCD (blue curves) or mock-treated (dashed
green curves) was isolated by affinity chromatography and SDS-PAGE prior to LC-MS.
Identified cross-linked peptides represent intra-molecular TatC contacts between S2 and E103
(top), quantified on the 2+ charged precursor observed at m/z 719.8681 (molecular mass of
1437.7212 Da), and between E244 and K101 (bottom), quantified on the 4+ charged precursor
at m/z 550.7523 (molecular mass of 2198.9788 Da). (b) Model of the E. coli TatC structure
adapted from the crystal structure of A. aeolicus TatC (PDB: 4B4A and 4HTT). Residues
involved in the DCCD-mediated TatC cross-links specified in (a) are marked in blue
(carboxylates) and yellow (amino groups). These intramolecular cross-links suggest an
orientation of the N- and C-termini as modelled here, in which both cytosolic tails fold back
towards the TM 2/TM 3 loop.
Fig. 1
6
5
4
3
2 1b
1a
Fig. 2
b
1 2 3 4 5 6
c
TatC
TatB
INV
TatABC
TatAC
wt TatC
E170A
DCCD - + - + - +
C
1b
4
2
N
1a
5
6
3
E170
cis
trans
αTatC
INV
TatABC
wt TatC
E170A
25 -
1 2
d
a
E4
D63
E244
D248
E103
INV TatABBpaC
F2 I4 F6
UV - + - + - +
DCCD
- + - + - +
p
TorA-
mCherry
(TmC)
55 -
70 -
35 -
40 -
TatB x TmC
TatC x TmC
100 -
1 2 3 4 5 6 7 8 9
INV TatABC
202Bpa
TatABC
206Bpa
TatABC
206Bpa TatABC
208Bpa
TatABC
9Bpa
TatC
E170A
UV - + - + - + - + - +
DCCD - + - + - + - + - +
55 -
35 -
40 -
c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
p
TorA-
mCherry
(TmC)
INV TatABC
206Bpa
CCCP
- +
UV - + - +
- 55
- 35
- 40
- 35
p
m
Fig. 3
16
17 18 19
TorA-
mCherry
b a
1a
1b
2
3
4
5
6
1a
1b
2
3
4
5
6
mature protein
RR
E170
E170
2
202
cis
trans
206
208
4
6
9
a
b
MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA/AQAATDAVEF
14 27
n-region h-region c-region
mature protein
consensus motif cleavage site
c
55 -
35 -
40 -
p TorA-mCherry
(TmC)
TatB x TmC ( )
TatC x TmC ( )
TmC F14Bpa L27Bpa
INV TatABC
UV - + - +
CCCP - + - +
TatA x TmC ( )
1 2 3 4 5 6
Fig. 4
TmC F14Bpa L27Bpa
INV TatABC
wt TatC E170A
wt TatC E170A
UV - + - + - + - +
DCCD
- +
- + - + - +
55 -
35 -
40 -
TatB x TmC ( )
TatC x TmC ( )
TatA x TmC ( )
1 2 3 4 5 6 7 8 9 10 11
12
p
TorA-mCherry
(TmC)
35 - p
m
TorA-mCherry
1 2 3 4 5 6 7 8 9 10 11 12
processing: 41% 36% 16%
INV TatABC TatAC
DMSO CCCP DCCD DMSO CCCP DCCD
PK - + - + - + - + - + - +
Fig. 5
INV TatACE170 A
DMSO CCCP DCCD
PK - + - + - +
p
m
TorA-mCherry
processing: 27%
27% 25%
a
b
1 2 3 4 5 6
35 -
25 -
25 -
Fig. 6
a
b
E244
K101
E103
N-terminus
Retention Time / min
18.5 19
Relative Intensity
100
0
100
0
Retention Time / min
24 24.5
m/z
719.8687
m/z
550.7523
Relative Intensity
