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Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide



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 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.
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,
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:
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 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.
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
(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
Glutamate 170 of E. coli TatC becomes
quantitatively modified by DCCD
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
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
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-
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
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).
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
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
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
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
Experimental Procedures
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
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%
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, (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
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).
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
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).
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
2 1b
Fig. 2
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... DCCD is a zero-space cross-linker forming isopeptide bonds between carboxyl and amino group-containing side chains of proteins (50). By means of a quantitative mass spectrometric analysis of DCCD-treated TatC, we identified distinct intramolecular contact sites of TatC suggesting that the N-terminal and C-terminal tails of the molecule can tightly pack against the transmembrane core of TatC (49). Here, we have extended this approach onto the entire TatBC complex based on the premise that novel TatB-TatB and TatB-TatC contacts, if revealed by treatment with DCCD, might allow new insights into the composition and functioning of TatBC complexes. ...
... Cross-linking of the E49, E53, and E77 residues of TatB to both the N-terminus of TatC and K239, located in the C-terminal domain of TatC, could reflect a close proximity of both ends of TatC. Experimental evidence for such a conformation is provided by the intramolecular TatC cross-link that DCCD formed between E244 and S2 (Fig. 3A) and by our previous analysis (49). Of note, the DCCD-mediated cross-links between the Nterminal TatC S2 and the C-terminal TatC E244 were only obtained from monomeric TatC (Fig. 3A) indicating that within a TatBC complex, both surface-exposed ends of each TatC monomer in fact touch each other and do not swing out to connect with neighboring TatC protomers. ...
... When testing this, we, however, had to exclude alternative inhibitory effects of DCCD. As previously shown (49), DCCD modifies E170 of TatC and thereby interferes with the hair-pin insertion of a Tat signal peptide into the TatBC-binding cavity. The defective insertion is, however, largely prevented by using membrane vesicles that carry the TatC E170A variant. ...
Full-text available
Twin-arginine-dependent translocases transport folded proteins across bacterial, archaeal and chloroplast membranes. Upon substrate binding they assemble from hexahelical TatC and single-spanning TatA and TatB membrane proteins. Although structural and functional details of individual Tat subunits have previously been reported, the sequence and dynamics of Tat translocase assembly remain to be determined. Employing the zero-space cross-linker N,N'-dicyclohexylcarbodiimide (DCCD) in combination with LC-MS/MS, we identified as yet unknown intra- and intermolecular contact sites of TatB and TatC. In addition to their established intramembrane binding sites, both proteins were thus found to contact each other through the soluble N-terminus of TatC and the interhelical linker region around the conserved glutamyl residue Glu-49 of TatB from Escherichia coli. Functional analyses suggested that by interacting with the TatC N-terminus, TatB improves the formation of a proficient substrate-recognition site of TatC. The Glu-49 region of TatB was found also to contact distinct downstream sites of a neighboring TatB molecule and to thereby mediate oligomerization of TatB within the TatBC receptor complex. Finally, we show that global DCCD-mediated cross-linking of TatB and TatC in membrane vesicles or, alternatively, creating covalently linked TatC oligomers prevents TatA from occupying a position close to the TatBC-bound substrate. Collectively, our results are consistent with a circular arrangement of the TatB and TatC units within the TatBC receptor complex and with TatA entering the interior TatBC-binding cavity through lateral gates between TatBC protomers.
... Because the TatA oligomer is released by protonophore treatment of the TatA Q8A variant, we can infer that the connection between the TatA oligomer and the receptor complex still requires stabilization by the PMF until at least the polar cluster exchange step in the cycle. Residue E170 in TatC has previously been inferred to interact with either the substrate signal peptide or with TatA (Aldridge et al., 2014;Berks et al., 2014;Blummel et al., 2017;Ramasamy et al., 2013;Rollauer et al., 2012). These proposals are not invalidated by our observation that a TatC E170A variant is assembly-locked. ...
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The twin‐arginine protein translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of chloroplasts. The Tat translocation site is transiently assembled by the recruitment of multiple TatA proteins to a substrate‐activated TatBC receptor complex in a process requiring the protonmotive force. The ephemeral nature of the Tat translocation site has so far precluded its isolation. We now report that detergent solubilization of membranes during active transport allows the recovery of receptor complexes that are associated with elevated levels of TatA. We apply this biochemical analysis in combination with live cell fluorescence imaging to Tat systems trapped in the assembled state. We resolve sub‐steps in the Tat translocation cycle and infer that TatA assembly precedes the functional interaction of TatA with a polar cluster site on TatC. We observe that dissipation of the protonmotive force releases TatA oligomers from the assembled translocation site demonstrating that the stability of the TatA oligomer does not depend on binding to the receptor complex and implying that the TatA oligomer is assembled at the periphery of the receptor complex. This work provides new insight into the Tat transport cycle and advances efforts to isolate the active Tat translocon. The Tat translocon transports folded proteins via a unique and enigmatic mechanism. The translocon assembles transiently, only in the presence of a substrate, a protonmotive force, and only at native expression levels, and for this reason is exceedingly difficult to access experimentally. The recovery of a partially assembled Tat apparatus described here validates previous work with fluorescent fusion proteins, provides new insight into the transport cycle, and is a significant advance in our efforts to isolate the active translocon.
... Translocation is initiated when a substrate protein with the correct signal peptide is recognized and bound to the docking complex located in the membrane ( Figure 10). As soon as it binds, the TatC component of the docking complex inserts the substrate proteins into the membrane (Blümmel et al. 2017). This binding event triggers the proton motor force (PMF) and, consequently, the recruitment of pore-forming TatA protomers from a pool in the membrane to form the active TatABC-containing translocation site (Sargent 2007;Alcock et al. 2016;Wojnowska et al. 2018). ...
We have identified a membrane protein complex of Bacillus subtilis involving an unknown protein, YteJ, and SppA, a membrane protein first described as a signal peptide peptidase and later shown to be also involved in the resistance to antibacterial peptides of the lantibiotic family. Using deletion mutant strains, we showed that both proteins are involved in this resistance. In the ΔsppA strain, the ectopic overexpression of SppA not only restored the resistance, it also induced the formation of elongated cells, a phenotype suppressed by the simultaneous overexpression of YteJ. Furthermore, the expression of truncated versions of YteJ pinpointed the inhibitory role of a specific domain of YteJ. Finally, in vitro biochemical studies showed that SppA protease activity was strongly reduced by the presence of YteJ, supporting the hypothesis of an inhibition by YteJ. Our in vivo and in vitro studies showed that YteJ, via one of its domain, acts as a negative regulator of the protease activity of SppA in this complex. In conclusion, we have shown that SppA/YteJ complex is involved in lantibiotic resistance through the protease activity of SppA, which is regulated by YteJ.
... TatA and TatE share similar functions, whereas TatB is functionally distinct. The second protein family consists of a hydrophobic TatC subunit, which is a polytopic transmembrane protein and forms a precomplex with the TatB subunits [12][13][14]. In chloroplasts and proteobacteria, the TAT pathway consists of TatA, TatB, and TatC. ...
Twin arginine translocation (TAT) pathways have been extensively studied in bacteria and chloroplasts for their role in membrane translocation of folded proteins. However, an increasing number of organisms have been found to contain mitochondria-located TAT subunits, including plant mitochondria, which contain TAT subunits, though in an unusual arrangement with only TatB and TatC subunits. To date, no confirmed function has been attributed to mitochondrial TAT pathways in any organism. Using a truncation mutant approach, we demonstrate that the plant mitochondrial TatB (MTTATB) is required for complex III biogenesis. More specifically, MTTATB performs at a late stage in complex III biogenesis, conveying the translocation of the C terminus of the Rieske FeS subunit back across the inner membrane. This work confirms that plant mitochondria retained a functional TAT pathway for the Rieske FeS translocation, most likely from the original mitochondrial ancestor. It is hypothesized that the original mitochondria contained a bacteria-derived TAT pathway required for at least the Rieske FeS translocation. In several eukaryotic lineages, this mitochondrial TAT pathway was lost and replaced by BCS1. Interestingly, plant mitochondria appear to assemble complex III in the same subunit order as yeast and mammals but in contrast use bacteria-like assembly factors for this process.
... Together, they take the shape of a baseball glove or cupped hand with very restricted structural flexibility [64]. Notably, a conserved Glu residue (Glu170 in E. coli) is positioned close to the signal peptide binding pocket in the plane of the membrane and potentially perturbs the bilayer structure [12,64,65]. Additional residues needed for TatC function reside in the cytoplasmic N-region and the first cytoplasmic loop [61,66]. ...
Full-text available
The twin-arginine protein translocation (Tat) system has been characterized in bacteria, archaea and the chloroplast thylakoidal membrane. This system is distinct from other protein transport systems with respect to two key features. Firstly, it accepts cargo proteins with an N-terminal signal peptide that carries the canonical twin-arginine motif, which is essential for transport. Second, the Tat system only accepts and translocates fully folded cargo proteins across the respective membrane. Here, we review the core essential features of folded protein transport via the bacterial Tat system, using the three-component TatABC system of Escherichia coli and the two-component TatAC systems of Bacillus subtilis as the main examples. In particular, we address features of twin-arginine signal peptides, the essential Tat components and how they assemble into different complexes, mechanistic features and energetics of Tat-dependent protein translocation, cytoplasmic chaperoning of Tat cargo proteins, and the remarkable proofreading capabilities of the Tat system. In doing so, we present the current state of our understanding of Tat-dependent protein translocation across biological membranes, which may serve as a lead for future investigations.
... In bacteria, the TAT machinery is built from two types of proteins. Polytopic TatC comprising six transmembrane domains (TMDs) is the largest TAT protein and serves as the main component of the docking complex [16][17][18][19]. Different bacterial species contain between one and three paralogous TatA-type proteins, called TatA, TatB and TatE, each of which has an N-terminal TMD and a C-terminal helical domain exposed to the cytoplasm [8,20]. ...
Full-text available
Background Bacteria and mitochondria contain translocases that function to transport proteins across or insert proteins into their inner and outer membranes. Extant mitochondria retain some bacterial-derived translocases but have lost others. While BamA and YidC were integrated into general mitochondrial protein transport pathways (as Sam50 and Oxa1), the inner membrane TAT translocase, which uniquely transports folded proteins across the membrane, was retained sporadically across the eukaryote tree. Results We have identified mitochondrial TAT machinery in diverse eukaryotic lineages and define three different types of eukaryote-encoded TatABC-derived machineries (TatAC, TatBC and TatC-only). Here, we investigate TatAC and TatC-only machineries, which have not been studied previously. We show that mitochondria-encoded TatAC of the jakobid Andalucia godoyi represent the minimal functional pathway capable of substituting for the Escherichia coli TatABC complex and can transport at least one substrate. However, selected TatC-only machineries, from multiple eukaryotic lineages, were not capable of supporting the translocation of this substrate across the bacterial membrane. Despite the multiple losses of the TatC gene from the mitochondrial genome, the gene was never transferred to the cell nucleus. Although the major constraint preventing nuclear transfer of mitochondrial TatC is likely its high hydrophobicity, we show that in chloroplasts, such transfer of TatC was made possible due to modifications of the first transmembrane domain. Conclusions At its origin, mitochondria inherited three inner membrane translocases Sec, TAT and Oxa1 (YidC) from its bacterial ancestor. Our work shows for the first time that mitochondrial TAT has likely retained its unique function of transporting folded proteins at least in those few eukaryotes with TatA and TatC subunits encoded in the mitochondrial genome. However, mitochondria, in contrast to chloroplasts, abandoned the machinery multiple times in evolution. The overall lower hydrophobicity of the Oxa1 protein was likely the main reason why this translocase was nearly universally retained in mitochondrial biogenesis pathways. Electronic supplementary material The online version of this article (10.1186/s12915-018-0607-3) contains supplementary material, which is available to authorized users.
The Twin-Arginine Pathway for Protein Secretion, Page 1 of 2 Abstract About 20 to 30% of proteins synthesized in the bacterial cytoplasm are destined for extracytoplasmic locations ( 1 ). They pass the cytoplasmic membrane using specialized transport systems, involving gated pores, energy, and signal peptides to direct protein export. Two major protein export systems are known, namely, the general secretory (Sec) pathway and the twin-arginine translocation (Tat) pathway ( Fig. 1 ). Most proteins use the Sec pathway, common to all domains of life. The Tat pathway, the focus of this review, is more exclusive. For example, it has only ∼30 native substrates in the Gram-negative bacterium Escherichia coli, and it is not universally conserved ( 2 ).
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The twin arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in their binding to the Tat translocase, but some facets of this interaction remain unclear. Here, we investigated the role of the hydrophobic (h-) region of the Escherichia coli TMAO reductase (TorA) signal peptide in TatBC receptor binding in vivo and in vitro. We show that besides the RR motif, a minimal functional h-region in the signal peptide is required for Tat-dependent export in E. coli. Furthermore, we identified mutations in the h-region that synergistically suppressed the export defect of a TorA[KQ]-30aa-MalE Tat reporter protein in which the RR motif was replaced with a lysine-glutamine pair. Strikingly, all suppressor mutations increased the hydrophobicity of the h-region. By systematically replacing a neutral residue in the h-region with various amino acids, we detected a positive correlation between the hydrophobicity of the h-region and the translocation efficiency of the resulting reporter variants. In vitro crosslinking of residues located in the periplasmically -oriented part of the TatBC receptor to TorA[KQ]-30aa-MalE reporter variants harboring a more hydrophobic h-region in their signal peptides confirmed that unlike in TorA[KQ]-30aa-MalE with an unaltered h-region, the mutated reporters moved deep into the TatBC -binding cavity. Our results clearly indicate that, besides the Tat motif, the h-region of Tat signal peptides is another important binding determinant that significantly contributes to the productive interaction of Tat precursor proteins with the TatBC receptor complex.
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The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast which were differentiated into myotubes followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we analyzed changes in the relative abundance of 2,588 proteins, of which 387 were significantly regulated in myoblasts versus myotubes. Changes in protein expression generally reflected the drastic phenotypic conversion of the cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Since an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an exemplary fashion, we analyzed the actin-binding multi-adaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our dataset provides an invaluable resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics and mobility that will greatly advance our understanding of Z disc dynamics and signaling.
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Twin-arginine translocation (Tat) systems mediate the transmembrane translocation of completely folded proteins that possess a conserved twin-arginine (RR) motif in their signal sequences. Many Tat systems consist of three essential membrane components named TatA, TatB, and TatC. It is not understood why some bacteria, in addition, constitutively express a functional paralog of TatA called TatE. Here we show, in live Escherichia coli cells, that, upon expression of a Tat substrate protein, fluorescently labeled TatE-GFP relocates from a rather uniform distribution in the plasma membrane into a number of discrete clusters. Clustering strictly required an intact RR signal peptide and the presence of the TatABC subunits, suggesting that TatE-GFP associates with functional Tat translocases. In support of this notion, site-specific photo cross-linking revealed interactions of TatE with TatA, TatB, and TatC. The same approach also disclosed a pronounced tendency of TatE and TatA to hetero-oligomerize. Under in vitro conditions, we found that TatE replaces TatA inefficiently. Our collective results are consistent with TatE being a regular constituent of the Tat translocase in E. coli.
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The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is comprised of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate-bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild type tatC. Single substitutions that confer dominant negative TatC activity localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue-native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate-induced disulphide crosslink in transmembrane helix five of TatC. Our findings show that TatC functions as an obligate oligomer. This article is protected by copyright. All rights reserved.
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The so-called Tat (twin-arginine translocation) system transports completely folded proteins across cellular membranes of archaea, prokaryotes and plant chloroplasts. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures. We have now elucidated the fine architecture of TatBC oligomers assembled to form closed intramembrane substrate-binding cavities. The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers. We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors.
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The twin arginine translocase (Tat) transports folded proteins of widely varying size across ionically tight membranes with only 2-3 components of machinery and the protonmotive force. Tat operates by a cycle in which the receptor complex combines with the pore-forming component to assemble a new translocase for each substrate. Recent data on component and substrate organization in the receptor complex and on the structure of the pore complex inform models for translocase assembly and translocation. A translocation mechanism involving local transient bilayer rupture is discussed. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.
Significance The twin-arginine translocation (Tat) system transports folded proteins across the prokaryotic inner membrane and the thylakoid membrane of plant chloroplasts. Proteins are targeted to the Tat system by signal peptides containing a highly conserved twin arginine motif. We isolated suppressors in the TatB component that allowed a Tat substrate with a defective twin arginine motif to be transported. The strongest of these suppressors, TatB F13Y, resulted in the constitutive assembly of the Tat translocase in the absence of signal peptide binding. These results show that Tat signal peptides have two separable roles: they target their passenger proteins to the Tat machinery but they also trigger the assembly of the active Tat transporter.
MaxQuant is one of the most frequently used platforms for mass-spectrometry (MS)-based proteomics data analysis. Since its first release in 2008, it has grown substantially in functionality and can be used in conjunction with more MS platforms. Here we present an updated protocol covering the most important basic computational workflows, including those designed for quantitative label-free proteomics, MS1-level labeling and isobaric labeling techniques. This protocol presents a complete description of the parameters used in MaxQuant, as well as of the configuration options of its integrated search engine, Andromeda. This protocol update describes an adaptation of an existing protocol that substantially modifies the technique. Important concepts of shotgun proteomics and their implementation in MaxQuant are briefly reviewed, including different quantification strategies and the control of false-discovery rates (FDRs), as well as the analysis of post-translational modifications (PTMs). The MaxQuant output tables, which contain information about quantification of proteins and PTMs, are explained in detail. Furthermore, we provide a short version of the workflow that is applicable to data sets with simple and standard experimental designs. The MaxQuant algorithms are efficiently parallelized on multiple processors and scale well from desktop computers to servers with many cores. The software is written in C# and is freely available at
Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by peroxisomal targeting sequences (PTS). In the following, the assembled receptor-cargo complex is targeted to the peroxisomal membrane where it docks to the docking-complex as part of the peroxisomal translocation machinery. The docking-complex is composed of Pex13p, Pex14p and in yeast also Pex17p, whose function is still elusive. In order to characterize the function of Pex17p, we compared the composition and size of peroxisomal receptor-docking complexes from wild-type and pex17Δ cells. Our data demonstrate that the deficiency of Pex17p affects the stoichiometry of the constituents of an isolated 600 kDa complex and that pex17Δ cells lack a high molecular weight complex (> 900 kDa) of unknown function. We identified the dynein light chain protein Dyn2p as additional core component of the Pex14p/Pex17p-complex. Both, Pex14p and Pex17p interact directly with Dyn2p, but in vivo, Pex17p turned out to be prerequisite for an association of Dyn2p with Pex14p. Finally, like pex17Δ also dyn2Δ cells lack the high molecular weight complex. As dyn2Δ cells also display reduced peroxisomal function, our data indicate that Dyn2p-dependent formation of the high molecular weight Pex14p-complex is required to maintain peroxisomal function on wild-type level.