From the Institute for Virus Research, Kyoto
University, Kyoto 606-8507, Japan, the § Graduate School of
Science, Osaka University, Toyonaka, Osaka 560-0043, Japan, the
¶ Graduate School of Science, University of Tokyo, Tokyo
113-0033, Japan, the
Departments of Medical Biochemistry and
Genetics, of Chemistry, and of Biochemistry and Biophysics, Texas
A&M University, College Station, Texas 77843, and the
** Biomolecular Engineering Research Institute, Suita,
Osaka 565-0874, Japan
Received for publication, January 9, 2003
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ABSTRACT |
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SecY and SecE are the two principal
translocase subunits that create a channel-like pathway for the transit
of preprotein across the bacterial cytoplasmic membrane. Here we report
the cloning, expression, and purification of the SecYE complex
(TSecYE) from a thermophilic bacterium, Thermus
thermophilus HB8. Purified TSecYE can be reconstituted into
proteoliposomes that function in T. thermophilus SecA
(TSecA) dependent preprotein translocation. After the mixing of
TSecYE derivatives labeled with either a donor or an acceptor
fluorophore during reconstitution, fluorescence resonance energy
transfer experiments demonstrated that 2 or more units of TSecYE in the
lipid bilayer associate to form a largely non-exchangeable oligomeric structure.
During the course of biogenesis some proteins must traverse a
membrane. The cytoplasmic (inner or plasma) membrane of prokaryotic cells and the endoplasmic reticulum membrane of eukaryotic cells are
equipped with conserved protein complexes called the SecYE complex,
Sec61 complex, translocase, or translocon. These complexes are thought
to serve as a pathway (channel) for protein translocation. The
Escherichia coli SecYEG complex, one of the most
intensively characterized translocon components (1), functions in
conjunction with the SecA ATPase to drive protein movement through the
prokaryotic cytoplasmic membrane (2). SecY and SecE are integral
membrane proteins with 10 and 3 transmembrane segments, respectively,
and they are sufficient to reconstitute basic
SecA-dependent preprotein translocation activity (3).
Structural biology studies of the Sec translocase have begun to yield
some useful information (4-7). Electron microscopic images suggest
that the SecYEG complex indeed forms a ringlike structure with a hole
at the center. It has been estimated that 2-4 units of the
SecYE(G) complex constitute a channel. However, the exact
subunit structure of the translocation channel has not been elucidated.
A biochemical study of a SecYEG complex that bears a preprotein
translocation intermediate suggests that an active translocase complex
contains only one SecYEG heterotrimer (8). Another study used an
electrophoretic approach to show that an active translocation channel
contains two SecYEG heterotrimers (9). Dimeric SecYEG has also been
observed in the first three-dimensional image obtained for a translocon
(7). The quaternary structure of the SecYEG complex is an important
unsolved question.
Spectrofluorometry provides a useful means to study the dynamic nature
of proteins, including those integrated into membranes (10). We were
thus prompted to apply the
FRET1 technique to
characterize protein translocase components. Our experience indicates
that the E. coli SecYE(G) complex is not ideal for
physicochemical characterization because of its instability, insolubility, and other characteristics of intractability. As an
alternative model organism for the study of protein translocase, we
became interested in Thermus thermophilus HB8 (11). In this article we report on the initial characterization of the T. thermophilus SecYE complex (TSecYE) with special reference to its
quaternary structure as studied by FRET approaches. We show that TSecYE
forms a constitutive oligomer in the membrane.
Bacterial Strains--
TSecYE was expressed in E. coli host strain AD202 (MC4100,
ompT::kan; Ref. 12), whereas
YccA-His6-Myc-Cys (see below) was expressed in AD1672, a
Cloning of SecY and SecE from T. thermophilus HB8 and
Construction of a TSecYE Expression System--
The secY
and secE genes were amplified from T. thermophilus HB8 genomic DNA (purchased from Takara Shuzo)
using Pfx DNA polymerase (Invitrogen) and the
following PCR primers, designed on the basis of the genomic sequence of
this bacterium (GenBankTM) accession numbers
AB086887 and AB086886, for secY and secE,
respectively). The primers for secY were
5'-CTGCCATGGTCAAGGCCTTCTGGAGCGCC-3' and
5'-GACTCTAGACTAATGGTGATGGTGATGGTGCCGGTTCCGCCCCCGCAGGC-3', in which the underlined letters represent NcoI
(upstream primer) and XbaI (downstream primer) recognition
sequences introduced for the purpose of cloning. The upstream primer
was designed such that the TSecY coding region can be fused in frame to
the second codon of lacZ Introduction of the A51C Mutation into TSecY--
A plasmid
encoding TSecYE with an A51C alteration in SecY was named pHM497. The
QuikChange method (Stratagene) was used with an appropriate
oligonucleotide primer to introduce this mutation, and a DNA fragment
with a confirmed sequence alteration was cloned back into pTT008.
Construction of a Plasmid Encoding
YccA-His6-Myc-Cys--
Plasmid pKH303 encoding
YccA-His6-Myc (17) was used as a template for site-directed
mutagenesis to attach Cys to the C terminus of
YccA-His6-Myc. A modified QuikChange method (18) was used with an appropriate oligonucleotide primer. The resulting plasmid of
confirmed DNA sequence was named pHM551.
Purification of TSecYE Complex--
Cells of strain AD202 that
carried both pTT008 and pSTD343
(lacIq)2
were grown at 40 °C overnight in L medium (10 g of Bacto-trypton, 5 g of yeast extract, and 5 g of NaCO/liter) supplemented with ampicillin
(50 µg/ml) and chloramphenicol (20 µg/ml). These uninduced conditions proved optimal for accumulation of the TSecYE complex; induction was followed by rapid cell lysis. The total membrane fraction
was prepared as described previously (19). The membrane was solubilized
with 2.5% (w/v) N-octyl- Purification of YccA-His6-Myc-Cys--
Strain AD1672
carrying pHM511 (YccA-His6-Myc-Cys) was precultured at
37 °C overnight in 200 ml of terrific broth (12 g of Bacto-trypton, 24 g of yeast extract, 4 ml of glycerol, and 72 mmol
KH2PO4/liter) supplemented with 0.4% glucose and
ampicillin (50 µg/ml) and then inoculated into 2 liters of L medium
containing ampicillin (50 µg/ml),
isopropyl-1-thio- Reconstitution of TSecYE Proteoliposomes and Activity
Assays--
The purified TSecYE complex was mixed with a 4000-fold
molar excess of E. coli phospholipids (or synthetic
phospholipids of phosphatidylglycerol:phosphatidyl ethanolamine = 3:7 (w/w)) in buffer C. After incubation on ice for 30 min, the
solution was mixed with SM2 beads (Bio-Rad), pre-equilibrated with 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2,
50 mM KCl, and 10% (w/v) glycerol, and incubated at
4 °C for 2 h with mild mixing (300 rpm). The solution was
transferred to fresh buffer-equilibrated SM2 beads and incubated
overnight to further absorb the detergent. The resulting turbid
solution was ultracentrifuged to recover proteoliposomes, which were
resuspended in 50 mM Tris-HCl (pH 7.5) and 50 mM KCl and stored at
In vitro preprotein translocation assays were carried out by
the procedures described previously (20, 21) using either inverted
membrane vesicles from the TSecYE-overproducing strain or TSecYE
proteoliposomes and SecA from T. thermophilus HB8 (TSecA), which will be described
elsewhere.3 The ATPase
activity of TSecA was assayed using published procedures (22).
Preparation of 5-Iodoacetamidefluorescein- and
Tetramethylrhodamine-5-iodoacetamide-labeled TSecYE Complexes--
A
purified preparation of the TSecY(A51C)E complex in buffer C containing
25 µM Tris (2-carboxyethyl) phosphine hydrochloride was
mixed with a 10-fold molar excess of 5-iodoacetamidefluorescein ( Preparation of
Tetramethylrhodamine-5-iodoacetamide-labeled
YccA- His6-Myc-Cys
(YccA(Rh))--
YccA-His6-Myc-Cys was labeled with
tetramethylrhodamine-5-iodoacetamide as described above. After the
removal of free fluorescent reagent by a Hi-trap desalting column, the
sample was diluted 10-fold with buffer B and purified further by
Hi-trap Q-column chromatography. Labeling efficiency, as estimated by
SDS-PAGE mobility shift, was at least 80%.
Fluorescence Spectroscopy--
Spectrofluorometry was carried
out at 25 °C in 50 mM Tris-HCl (pH 7.5) and 50 mM KCl using a Hitachi F-4500 fluorometer. Fluorescein was
excited at 492 nm (5-nm bandpass), and its emission was scanned from
500 to 600 nm (5-nm bandpass, scan rate 240 nm/min). Data shown are an
average of three successive spectra. Rhodamine was excited at 543 nm
and scanned from 550 to 600 nm as described above. Fluorescence
emission spectra of the TSecYE preparations that were labeled with
donor (fluorescein) and acceptor (rhodamine) fluorophores were
recorded. To observe FRET after proteoliposome reconstitution,
reconstitution was carried out with several combinations of labeled
TSecYE. Typically, 80 pmol of TSecY(Fl51)E or TSecY(Rh51)E was mixed
with 800 µg of E. coli phospholipids for preparation of
proteoliposomes containing a labeled TSecYE species. To obtain proteoliposomes with both the donor dye- and the acceptor dye-labeled TSecYE species, 80 pmol of TSecY(Fl51)E and 240 pmol of TSecY(Rh51)E were subjected to reconstitution. For preparation of non-labeled TSecYE
proteoliposomes, 400 pmol of TSecY(A51C) was used for reconstitution. The proteoliposomes were resuspended in 80 µl of 50 mM
Tris-HCl (pH 7.5) and 50 mM KCl, and 5 µl of the
suspension was diluted with 995 µl of the same buffer for the
fluorescence measurement. The actual amount of TSecY(Fl51)E in the
assay solution (1 ml) was 1.5-2.5 pmol. As a control, reconstitution
was also carried out with a combination of TSecY(Fl51)E and YccA(Rh).
Flotation Centrifugation--
A 20-µl suspension of
reconstituted proteoliposomes was mixed with 80 µl of 48% (w/w)
sucrose in 3 mM EDTA (pH 7.0) and placed at the bottom of a
centrifugation tube on which 300 µl each of 48, 40, and 33% sucrose
and 400 µl of the EDTA medium alone were layered. The sample was
centrifuged at 40,000 rpm for 16 h at 4 °C in a Hitachi
S55S rotor.
Liposome Fusion by Treatment with PEG 3350--
PEG
3350-mediated liposome fusion was carried out as follows. A suspension
of liposomes was mixed with PEG 3350 (Sigma; final concentration of
12.5% (w/v)) and incubated at 37 °C for 5 min to induce liposome
fusion.4 The efficiency of
liposome fusion was assessed by the cancellation of FRET between
fluorophore-labeled phospholipids (23) and by dynamic light-scattering
changes (see below).
Measurement of Liposome Size by Dynamic Light
Scattering--
Size distribution profiles of proteoliposome vesicles
were determined by dynamic light-scattering measurement using an
FPAR-1000 fiber optics particle analyzer (Photal Otsuka Electronics)
and the CONTIN program according to the manufacturer's instructions.
Genomic Identification of SecY and SecE Components in T. thermophilus HB8--
The completed genome sequence of T. thermophilus HB8 indicates the existence of SecY and SecE homologs
in this bacterium. Fig. 1 shows their
deduced amino acid sequences. TSecY is homologous to E. coli
SecY with 44% identity and ~80% similarity in overall amino acid
sequences; two insertions and two gaps, relative to the E. coli protein, are notable (Fig. 1). TSecE differs more markedly in
overall architecture from E. coli SecE. It contains only one
putative transmembrane segment, a feature similar to that of
Bacillus subtilis SecE. The sequence
similarity at the C-terminal half of the cytoplasmic region, the most
conserved segment in SecE (24), was ~33%.
Purification of TSecYE--
The possible advantage of the
increased thermal stability of proteins from thermophilus prompted us
to characterize TSecYE with the ultimate objective of understanding its
structure. Plasmid pTT008 encodes TSecY-His6 and TSecE-Myc.
The calculated molecular weights of these products are 49,010 and
9,174, respectively. These proteins accumulated to compose
approximately 10% of the cytoplasmic membrane proteins. The TSecYE
complex was solubilized with 2.5% (w/v) octylglucoside and purified
successively by Ni-NTA affinity column chromatography, gel filtration
chromatography, and cation exchange chromatography. The Ni-NTA column
step also served to exchange the detergent from 1.25% (w/v)
octylglucoside to 0.2% (w/v) C12E8. The
complex was purified to apparent homogeneity, with only two components
detected upon SDS-PAGE (Fig. 2A,
lane 1). From a 10-liter culture we obtained 4 mg of
purified complex.
The complex formed a symmetrical peak of 120-130 kDa on gel
filtration (Fig. 2B). Assuming that a TSecYE heterodimer is
associated with one micelle of the detergent
C12E8, with a probable molecular mass of 66 kDa the detergent-complexed TSecYE will have an apparent molecular mass of approximately 124 kDa.5 The gel filtration result
is consistent with this value. Some preparations of TSecY(A51C)E
contained a minor product migrating slower than TSecY(A51C) on
non-reducing SDS-PAGE (Fig. 2C; the band shown as
(TSecYE)2). On gel filtration, this product was eluted
ahead of the main SecY(A51C)E peak at a position corresponding to
200-300 kDa (Fig. 2C). Because this species disappeared on Functional Reconstitution of TSecYE into Proteoliposomes--
The
purified TSecYE complex was mixed with E. coli
phospholipids, and the detergent concentration was lowered by
adsorption to SM2 beads. Efficient integration of the complex into the
artificial membrane was confirmed by flotation centrifugation
experiments (see Fig. 4). The translocation activity of TSecYE in
proteoliposomes was examined by incubation with urea-denatured E. coli proOmpA-His6-Myc, TSecA, and ATP at 50 °C.
During this incubation, proOmpA acquired resistance to externally added
proteinase K (Fig. 3A,
lanes 1-4), which was lost after solubilization of
proteoliposomes with Triton X-100 (Fig. 3B, lane
6). Omission of ATP or TSecA abolished the translocation activity
(Fig. 3B, lanes 4 and 5). The
translocation activity was temperature-dependent; it was
undetectable at 0 °C (Fig. 3B, lane 1) and was two times
higher at 50 °C than at 37 °C (Fig. 3B, lanes
2 and 3). E. coli SecYEG complex did not
work at 50 °C (Fig. 3C, lower panel,
lane 5) and E. coli SecA did not substitute for
TSecA for proOmpA translocation into the TSecYE proteoliposome at any
temperature examined (Fig. 3C, lane 2), eliminating the possibility that the activity was caused by
contaminating E. coli components. Conversely, TSecA did not
function in combination with E. coli SecYEG (Fig.
3C, lane 4).
The ATPase activity of TSecA as assayed at 60 °C was negligible in
the absence of membranes, but it was stimulated markedly by the
addition of inverted membrane vesicles from the TSecYE-overproducing strain (Fig. 3D). ProOmpA further stimulated the ATPase
activity, although the extent of stimulation by this E. coli
preprotein was limited. These results, taken together, indicate that
the recombinant TSecYE complex is functional when incorporated into an
E. coli phospholipid bilayer.
Characterization of TSecYE Proteoliposomes for Use in FRET
Analysis--
Although a monomeric TSecYE heterodimer is the major
form that TSecYE assumes in detergent, we wanted to know the oligomeric state of the SecYE complex after integration into the lipid bilayer. To
address this question, we used a FRET approach. TSecYE lacks cysteine.
We replaced Ala51 of TSecY in the first periplasmic region
with cysteine. The mutant TSecY(A51C)E complex was purified,
reconstituted into proteoliposomes (Fig.
4A), and found to be active in
TSecA-dependent translocation of proOmpA (Fig.
5A). The mutant TSecYE was
modified with either of two different thiol-reactive iodoacetamide
derivatives, 5-iodoacetamidofluorescein or
tetramethylrhodamine-5-iodoacetamide, to yield fluorescein-labeled TSecY(Fl51)E or rhodamine-labeled TSecY(Rh51)E. Absorption measurements showed that TSecY(A51C)E was modified almost quantitatively with these
compounds (data not shown). It was noted that among five TSecY mutants
with a unique Cys residue in each periplasmic loop (at
Ala51, Gly146, Leu206,
Gly299, or Thr389), TSecY(A51C) gave the
highest labeling efficiency. Thus, all of the following fluorescence
experiments were carried out using this TSecY derivative.
Flotation centrifugation showed that SecYE was efficiently
reconstituted into proteoliposomes, even after modification with fluorescein or rhodamine (Fig. 4, B and C).
Reconstitution was also successful with a mixture of TSecY(Fl51)E and
TSecY(Rh51)E (Fig. 4D). Light-scattering analysis revealed
that our procedures produced homogeneously populated vesicles of a mean
diameter of 200-300 nm (see Fig. 7B, squares,
for TSecY(Fl51)E-containing proteoliposomes).
The reconstituted proteoliposomes carrying either TSecY(Fl51)E,
TSecY(Rh51)E, or both were fully active in TSecA- dependent translocation of proOmpA (Fig. 5, B-D). These
results indicate that the TSecYE complex can effectively be
integrated into the E. coli phospholipid bilayer in
vitro, generating active and homogenous vesicles. In addition, the
fluorophore modification does not interfere with the assembly or
function of the TSecYE translocase.
FRET Analysis of the Oligomeric Status of TSecYE--
The FRET
phenomenon results from the transfer of excited-state energy from one
dye (the donor; here, fluorescein) to a second dye (the acceptor; here,
rhodamine) without the emission of a photon. The efficiency of FRET
depends on, among other things, the distance between the donor and
acceptor dyes. For the fluorescein-rhodamine pair, FRET will be
reliably detected only if the dyes are separated by less than 80 Å (25, 26). Although this could be achieved by neighboring but
non-interacting molecules if the membrane surface per one SecYE
molecule averages less than 6400 Å2, the use of a
1000-fold excess of phospholipid molecules (surface area, ~70
Å2/mol) in our experiment significantly precludes
such nonspecific FRET. Thus, FRET will be observed only if a
TSecY(Fl51)E and a TSecY(Rh51)E are co-localized as subunits of the
same translocase complex (25).
To evaluate the oligomeric state of the TSecYE complex after its
incorporation into the phospholipid bilayer, we used reconstituted proteoliposomes characterized above. Liposomes without any added protein, as well as proteoliposomes reconstituted with unlabeled TSecYE
proteins, showed no significant emission at 500-600 nm when they were
excited at 492 (data not shown). When proteoliposomes reconstituted with TSecY(Fl51)E were excited at 492 nm, a typical fluorescein emission spectrum was observed with a maximum at 518 nm
(Fig. 6A, black).
As expected, no fluorescein emission was detected when TSecY(Rh51)E
proteoliposomes were excited at 492 nm, but a small amount of direct
excitation of the rhodamine dye was present at 492 nm (Fig.
6A, pale blue). Also as expected, excitation of
TSecY(Rh51)E proteoliposomes at 543 nm exhibited rhodamine emission
with a peak at 572 nm (Fig. 6A, dark blue), whereas no rhodamine emission was observed with the fluorescein-labeled proteoliposomes (Fig. 6A, red). The fluorescence
intensities did not change when the vesicles were solubilized with
0.5% (w/v) Triton X-100, but the fluorescein emission was slightly
red-shifted and the rhodamine emission was slightly blue-shifted (data
not shown). Because the emission profiles of the two proteoliposomes were completely additive when mixed at the same concentrations (Fig.
6A, green and pink), the TSecYE
complexes in different proteoliposomes did not interact and no FRET
occurred between proteoliposomes.
We then reconstituted proteoliposomes with a mixture of the two labeled
TSecYE preparations (with a molar ratio of fluorescein- to
rhodamine-labeled protein of 1:3). In this case, we clearly observed
FRET between the two fluorophores. On excitation at 492 nm, the
intensity of the fluorescein peak (518 nm) decreased markedly (by
33%), whereas the rhodamine emission (572 nm) increased (Fig. 6B, black). This result should have represented
the association of the two differently labeled TSecYE species on
integration into the bilayer. The FRET efficiency was dependent
on the TSecY(Fl51)E to TSecY(Rh51)E ratio, because we observed a lower
extent of FRET with a 1:1 mixture of these components (data not shown).
On detergent solubilization of the proteoliposomes with either Triton
X-100 (Fig. 6B) or with C12E8 (data
not shown), the fluorescein emission increased, and the rhodamine
emission decreased to levels consistent with the absence of FRET
(compare the green spectra in Fig. 6, A and
B). This detergent-dependent elimination of FRET
demonstrates that the fluorescein- and rhodamine-labeled proteins had
associated in the membrane to form an oligomer of SecYE. Such an
association is the simplest scenario to explain the detection of FRET
in proteoliposomes. From theoretical consideration we have already
ruled out the alternative possibility that the membranes were too
crowded by the fluorescein- and rhodamine-labeled polypeptides.
Indeed, no significant change in fluorescence intensity was observed
when the FRET-positive proteoliposomes were fused with liposomes
containing no protein, thereby increasing further the average distance
between randomly distributed proteins in the bilayer (see below).
We also carried out a control experiment using an unrelated protein,
YccA, a membrane protein with seven transmembrane segments. Although
the cloned product, YccA-His6-Myc protein, contains two Cys
residues in the fourth transmembrane segment, they were not labeled
with tetramethylrhodamine-5-iodoacetamide. We constructed YccA-His6-Myc-Cys, in which a Cys was attached to the
periplasmically oriented C terminus of this protein.
YccA-His6-Myc-Cys was labeled with
tetramethylrhodamine-5-iodoacetamide and mixed 3:1 with
TSecY(Fl51)E for reconstitution into liposomes. Although the YccA
derivative was effectively incorporated into liposomes (data not
shown), no significant FRET from TSecY(Fl51)E to YccA(Rh) was
observed (Fig. 6C, black versus
green). Thus, the FRET we observed between the differently
labeled TSecYE complexes probably represented specific interaction
among the SecYE complexes.
The TSecYE Oligomer Subunits Do Not Exchange--
We also examined
whether the TSecYE subunits in a translocase oligomer are in an
association-dissociation equilibrium in the bilayer-integrated state.
Treatment of liposomes with 12.5% (w/v) PEG 3350 at 37 °C for 5 min
causes them to fuse.4 This result was confirmed here by two
independent experiments. First, liposomes containing
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled phosphatidylethanolamine and rhodamine-labeled phosphatidylethanolamine were mixed with non-labeled liposomes and treated with PEG 3350. This
resulted in increased average separation between donor and acceptor
dyes and in decreased FRET and, hence, in increased intensity of the
donor (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) fluorescence (Fig. 7A, column 1 versus column 2). Also, dynamic light-scattering data indicated that the PEG 3350 treatment increased the average size
of the reconstituted vesicles from 195.9 to 586.9 nm (Fig. 7B, open black squares versus open red
circles), although a marked broadening of the size distribution
curve was notable.
When proteoliposomes containing both TSecY(Fl51)E and TSecY(Rh51)E were
fused with those containing more than a 10-fold excess of unlabeled
TSecYE, no significant change in fluorescence intensity was observed
(Fig. 7C, green versus
blue). The FRET in either sample was completely abolished by
detergent treatment (black and red). Furthermore, fusion of
proteoliposomes containing TSecY(Fl51)E and those containing
TSecY(Rh51)E did not result in any detectable FRET (data not shown).
These results indicate that the FRET-eliciting association of SecYE
heterodimers results in an oligomeric complex that does not dissociate
in the time scale of minutes under the conditions of our experiment.
Thus, the SecYE heterodimer subunits in the assembled translocase in
the membrane appear to exchange very slowly with individual SecYE heterodimers.
The protein-translocating channel is a fundamental cellular
element that enables a considerable fraction of gene products to
achieve their subcellular localization. Thermophilic organisms provide
us with excellent opportunities to study the structure-function relationships of proteins because of the additional stability of their
protein products. However, not many membrane proteins have been
characterized from thermophiles. In this work, we cloned secY and secE genes from T. thermophilus and established a system to overexpress, purify, and
reconstitute the recombinant proteins TSecY and TSecE from E. coli. TSecYE expressed in E. coli as a membrane-integrated protein complex exhibited the in vitro
activity to mediate translocation of the E. coli proOmpA
protein into inverted membrane vesicles (data not shown). In a
detergent C12E8 solution, TSecY and TSecE form
a complex, TSecYE, that exists as a monomeric heterodimer. TSecYE is
sufficiently stable such that it can be purified to homogeneity after a
series of column chromatographies, and it can be reconstituted with
E. coli phospholipids into translocation-active proteoliposomes. This activity is dependent on TSecA and ATP, and its
optimal temperature is 50-60 °C. Although this is lower than the
optimal growth temperature of T. thermophilus, such a behavior is not unexpected, because the liposome vesicles were composed
of E. coli phospholipids, which might be unstable at higher
temperatures. On the other hand, the reconstituted proteoliposomes were
active at 37 °C, well below the minimum growth temperature of
T. thermophilus HB8 (~60 °C). Our attempts thus far
have failed to reconstitute active translocase with T. thermophilus phospholipids.
Despite the use of heterologous phospholipids, the reconstituted TSecYE
proteoliposomes exhibited absolute specificity to the cognate SecA
protein from the same organism. The intrinsic ATPase activity of TSecA
is extremely low (Fig.
3D).6 The ATPase
activity of TSecA is dramatically stimulated by membrane vesicles with
overproduced TSecYE. However, the addition of E. coli
proOmpA only moderately stimulated the TSecA ATPase activity. The
genomic sequence of T. thermophilus HB8 does not reveal an OmpA homologue in this organism,7
suggesting that this preprotein from E. coli is not an ideal substrate for the TSecA-TSecYE translocase. Our results showing that
only SecYE and SecA from the same organism can function as an
active translocase reinforces the notion that specific SecYE-SecA interaction is crucial for the protein translocase activity. Although the cytoplasmic regions of SecY are well conserved between T. thermophilus and E. coli (Fig. 1), both SecYE and
SecA could have specific determinants for the productive interaction.
The T. thermophilus-E. coli translocase
combination may prove useful for the study of specific interaction
between the SecA motor and the SecYE channel components.
Our results show that TSecYE exists as a monomer of the heterodimer
when in detergent-solubilized state. This "monomer" was the unit of
the modification with the fluorophore-bearing iodoacetamide derivatives, and the FRET observed in our reconstitution experiments represents a further association of this unit of the SecYE complex. Thus, our FRET results have shown that at least two TSecYE heterodimers form a stable complex in reconstituted proteoliposomes that are active
in supporting SecA-dependent preprotein translocation. These results are not consistent with the conclusion that only a single
SecYE heterodimer is sufficient to promote preprotein translocation on
the basis of biochemical studies of an E. coli SecYE complex
bearing a translocation intermediate (8).
Recent electron microscopic studies of B. subtilis or
E. coli SecYE(G) complex suggest that 2-4 SecYE(G)
complexes form a ring-shaped structure with a central "channel" of
15-50 Å (4, 5). However, the exact quaternary structure of the SecYEG
channel has not been determined. Manting et al. (5) proposed
a SecA-dependent formation of a tetrameric SecYEG complex.
More recently, Breyton et al. (7) revealed a dimeric
super-assembly of E. coli SecYEG by analysis of
two-dimensional crystals. We have now shown that TSecYE constitutively
forms an oligomer in functional proteoliposomes. Our FRET approach
complements the knowledge obtained by direct structural determination,
because it allows us to examine membrane-integrated and functional
SecYE complexes. It should be stressed, however, that our conclusion
does not mean that the translocation channel is static. Instead, it
presumably undergoes structural changes in response to SecA and
preprotein, probably accompanied by the opening/closing movements of a
gating device. More quantitative FRET analysis may prove useful as a
means to follow the dynamic nature of the SecYE translocation channel
during the initiation, continuation, and termination events of the
SecA-dependent translocation reaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ftsH sfhC21 derivative (13) of strain CU141 (14). Strains
JM109 and AD16 (15) were used for DNA manipulation experiments.
on the cloning vector by using
the NcoI site at this portion of lacZa. The
boldface G indicates a resulting mismatched base that resulted in a Leu
to Val change at the second position in the cloned TSecY. Italicized
bases were for a His6 tag attached to the C terminus. The
primers for secE were
5'-TCCTCTAGAAGGAGGTTTAAATTATGTTCGCCCGGCTGATC-3' and 5'-CTACTGCAGTCGCAGAAGCCCTATCAGG-3'. The upstream
primer contained the XbaI recognition sequence (underlined)
as well as an optimized E. coli SD sequence (boldface; Ref.
16). The downstream primer contained the PstI site
(underlined), and this resulted in an extension of Leu-Gln beyond the
natural C terminus, Arg60, of TSecE. In addition, an
oligonucleotide for a Myc epitope sequence (Fig. 1) flanked by the
PstI and HindIII restriction sites was used to
attach the Myc tag to the cloned TSecE C terminus. The
TSecY-His6, the TSecE, and the Myc DNA fragments were
inserted into appropriate restriction sites of a lac
promoter vector, pTV118N (Takara Shuzo), through several subcloning
steps. The resulting plasmid was named pTT008 and confirmed to carry a
DNA sequence for TSecY-His6-TSecE-Myc (with amino acid
changes indicated above) under the control of the lac promoter.
-D-glucopyranoside
in 50 mM Tris-HCl (pH 7.5), 2 mg/ml E. coli
phospholipids (Avanti), 20% (w/v) glycerol, and 0.1 mM
Pefabloc® (Merck) at 4 °C for 1 h with mild
mixing. After the removal of insoluble materials by
ultracentrifugation, supernatant was applied to a Ni-NTA column
equilibrated with buffer A consisting of 50 mM Tris-HCl (pH
7.5), 1.25% (w/v)
N-octyl-
-D-glucopyranoside, 0.5 mg/ml
E. coli phospholipids, and 20% (w/v) glycerol. The column was equilibrated with buffer B consisting of 50 mM Tris-HCl
(pH 7.5), 0.2% (w/v) C12E8, 300 mM
NaCl, and 20% (w/v) glycerol for detergent exchange. TSecYE was then
eluted with a linear 0-300 mM imidazole gradient in buffer
B. The TSecYE complex was stable in the C12E8
solution in the absence of phospholipids. Fractions containing the
TSecYE complex were pooled and concentrated with Ultrafree 4 (Millipore) for subsequent gel filtration chromatography with a HiLoad
16/60 Superdex 200 pg or Superose 6 (Amersham Biosciences) column
pre-equilibrated with buffer C (50 mM Tris-HCl (pH 8.1), 0.2% (w/v) C12E8, 300 mM NaCl, and
20% (w/v) glycerol). TSecYE complex was eluted in a single relatively
sharp peak. At this stage, the preparation was estimated by SDS-PAGE to
be at least 90% pure (Fig. 2A) and was used for activity
measurements. For FRET experiments, samples were further treated with a
desalting column and applied to a Hi-trap SP (Amersham Biosciences)
column that was eluted with a 0-0.5-M linear NaCl
gradient. The TSecYE complex was eluted at about 300 mM
NaCl. The TSecY(A51C)E mutant complex was also purified by the above
procedures. Protein concentration of the purified TSecYE complex was
determined with a micro-BCA protein assay kit (Pierce) using bovine
serum albumin as a standard. The molar extinction coefficient, at 280 nm, of TSecYE was determined to be 1.3 × 105
M
1 cm
1.
-D-galactopyranoside (1 mM) and cyclic AMP (1 mM) for further growth at
37 °C for 6 h. The YccA-His6-Myc-Cys protein was
partially purified by Ni-NTA column chromatography as described above
for TSecYE purification. The protein fractions, eluted at approximately
60 mM imidazole, were combined and passed through a Hi-trap
desalting column (Amersham Biosciences) pre-equilibrated with buffer D
(50 mM Tris-HCl (pH 8.1), 0.2% (w/v)
C12E8, and 20% (w/v) glycerol), followed by
Hi-trap Q (Amersham Biosciences) column chromatography with elution
with a 0-0.5-M linear NaCl gradient in buffer D. YccA-His6-Myc-Cys was eluted at approximately 200 mM NaCl as a preparation of approximately 90% purity.
80°C.
492 = 7.5 × 104
M
1 cm
1; Molecular Probes) or
tetramethylrhodamine-5-iodoacetamide (
543 = 8.7 × 104 M
1 cm
1;
Molecular Probes), dissolved in dimethyl sulfoxide, and incubated at
4 °C for 2 h with gentle mixing. Unreacted
fluorescent reagents were then quenched by incubation with 10 mM dithiothreitol for 10 min on ice and removed by passage
through a Hi-trap desalting column. The fluorescein- and
rhodamine-labeled proteins, designated TSecY(Fl51)E and TSecY(Rh51)E,
respectively, were concentrated and further purified by Hi-trap SP
column chromatography (see above). The elution profiles of
A280 and the dye absorbance coincided well for
the TSecY(A51C)E peak region. Absorbance measurements of the purified
proteins revealed that the TSecY(Fl51)E and TSecY(Rh51)E preparations
each contained one dye per protein on the basis of the molar extinction
coefficients of the protein and the dyes (see above).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequences of TSecY and TSecE.
Amino acid sequences of TSecY (A) and TSecE (B),
predicted from the T. thermophilus HB8 genomic sequence, are
compared with those from E. coli using the ClustalW program.
The transmembrane segments of E. coli proteins are
underlined. Some residue numbers are shown on the
right. Residues of identical, closely related, and similar
chemical characters between the two species are marked by
asterisks, double dots, and single
dots, respectively. Insertion and gaps are shown by
hyphens. The recombinant TSecY protein used in this study
carried a Leu to Val substitution introduced into the second position
(reversed). The His6 tag and Myc tag at the C terminus of
TSecY and TSecE, respectively, are also shown.
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Fig. 2.
Purification and oligomeric states of
detergent-solubilized TSecYE. A, electrophoretic
pattern of purified TSecYE. A sample of TSecYE (0.5 µg, left
lane) was analyzed by 10% PAGE using the SDS-Tricine system (27)
and Coomassie Brilliant Blue staining. B, gel
filtration profile. Purified TSecYE complex (20 µg) was applied to a
Superose 6 column, equilibrated with buffer C, and eluted with the same
buffer. Elution positions of the molecular weight markers are shown.
C, gel filtration of a TSecY(A51C)E preparation, which
included a minor oxidized component. A purified TSecY(A51C)E
preparation was treated on ice for 90 min with 5 mM
iodoacetamide and then applied to superose 6 column equilibrated with
buffer C containing 1 mM iodoacetamide. Each fraction was
analyzed by 10% SDS-PAGE (Laemmli system) without the inclusion of
-mercaptoethanol and subsequent anti-hexahistidine Western blotting.
The band indicated by (TSecYE)2 represents a
disulfide-bonded dimer of TSecY(A51C)E, which was generated presumably
by air oxidation during purification. Note that TSecY migrated
differently in the two PAGE systems shown in A and
C and that there was some distortion of the band (at
fractions corresponding to volume of 16-17 ml) that was introduced
during the blotting procedures.
-mercaptoethanol treatment (data not shown), it must have
represented a disulfide-bonded dimer of TSecY. Thus, the major TSecYE
peak corresponded to a monomeric form of this heterodimer. These
results strongly suggest that the TSecYE complex exists primarily as a (TSecY-TSecE)1 form in the C12E8
solution, although an equilibrium between 2(TSecYE)1 and
(TSecYE)2 may also occur to some extent.
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Fig. 3.
In vitro activities of protein
translocase from T. themophilus. A,
the time course of proOmpA translocation mediated by reconstituted
proteoliposomes. Reconstituted proteoliposomes (estimated SecYE content
of 0.2 µg) were subjected to a proOmpA-His6-Myc (33 ng)
translocation assay in the presence of ATP (5 mM) and TSecA
(20 µg/ml). Incubations proceeded at 50 °C for the indicated
periods, followed by proteinase K treatment (0.1 mg/ml for 20 min on
ice). After SDS-PAGE, proOmpA-His6-Myc was detected by
anti-Myc immunoblotting. The translocation yield was about 20% at 60 min. B, translocation requirements. Proteoliposomes
were subjected to the translocation assay for 60 min as described above
except for the omission of a component or a change in other reaction
conditions as indicated. C, SecA requirement of
translocation. The TSecYE proteoliposomes and the E. coli
SecYEG proteoliposomes (20) as indicated were subjected to the proOmpA
translocation assay in the presence of SecA from either T. thermophilus (T) or E. coli (E).
Incubation was carried out at either 37 °C or at 50 °C as
indicated for 60 min. D, TSecA ATPase activity.
TSecA (7 µg) was assayed for ATPase activity under the
conditions indicated. Inverted membrane vesicles prepared from
TSecYE-overproducing cells (20 µg protein) and urea-denatured
proOmpA-His6-Myc (0.3 µg) were included as specified. The
average of three measurements is shown with S.D. indicated by
horizontal bars. The background liberation of inorganic
phosphate in the absence of TSecA was subtracted.
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Fig. 4.
Proteoliposome formation with
fluorophore-labeled and unlabeled TSecYE. Proteoliposome samples
prepared with TSecY(A51C)E (A), TSecY(Fl51)E (B),
TSecY(Rh51)E (C), or a 1:1 mixture of TSecY(Fl51)E and
TSecY(Rh51)E (D) were subjected to flotation centrifugation
as described under "Materials and Methods." The step gradient was
fractionated into five fractions from top (left) to bottom
(right). The TSecY of each fraction was visualized by
SDS-PAGE and anti-hexahistidine immunoblotting.
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Fig. 5.
Fluorophore-labeled TSecYE Proteoliposomes
are functional. In vitro proOmpA translocation assays
were carried out using proteoliposomes reconstituted with
TSecY(A51C)E (A), TSecY(Fl51)E (B),
TSecY(Rh51)E (C), or a 1:1 mixture of
TSecY(Fl51)E and TSecY(Rh51)E (D), as described in the
legend to Fig. 3A. After incubation for the indicated
periods, protease-resistant proOmpA-His6-Myc was detected
by Western blotting.
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Fig. 6.
Fluorescence analysis of the TSecYE
complex in reconstituted proteoliposomes. A,
fluorescence spectra of proteoliposomes containing TSecY(Fl51)E or
TSecY(Rh51)E. Proteoliposomes containing TSecY(Fl51)E were excited at
492 or 543 nm to obtain the emission spectra shown in black
or red, respectively. Emission spectra of proteoliposomes
containing TSecY(Rh51)E are also shown (excitation at 492 nm,
pale blue; 543 nm, dark blue), as well as those
for a 1:1 mixture of the above two samples (492 nm, green;
543 nm, pink). B, FRET after mixed
reconstitution. Emission spectra of proteoliposomes reconstituted with
a 1:3 mixture of TSecY(Fl51)E and TSecY(Rh51)E that were excited at 492 nm (black) or at 543 nm (red) are shown. The same
proteoliposomes were then solubilized with 0.5% (w/v) Triton X-100
before the emission scans were repeated (492 nm, green; 543 nm, blue). Background emission by liposomes without protein
was subtracted from each curve. C, lack of FRET between
TSecYE and YccA. Fluorescence emission spectra were recorded for
proteoliposomes reconstituted from a 1:3 mixture of TSecY(Fl51)E and
YccA(Rh) with excitation at 492 nm (black) or at 543 nm
(red). The same proteoliposomes were then solubilized with
0.5% (w/v) Triton X-100, and emission scans were repeated (492 nm,
green; 543 nm, blue). Background emission by
liposomes without protein was subtracted from each curve.
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Fig. 7.
Effects of proteoliposome fusion on FRET
observed with reconstituted TSecYE. A, PEG 3350-induced
liposome fusion as revealed by the disappearance of FRET between
phospholipid-attached fluorophores. Liposomes were prepared in the
presence of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled
phosphatidylethanolamine and rhodamine-labeled phosphatidylethanolamine
that were mixed with 125-fold weight excess of E. coli
phospholipids. They were then mixed with unlabeled liposomes at a
weight ratio of 1:3. The mixture was incubated with (columns
1 and 3) or without (column 2) PEG 3350 (final concentration of 12.5% (w/v)) at 37 °C for 5 min.
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (donor) fluorescence
emission at 530 nm was then measured using an excitation wavelength of
463 nm. Emission intensity in the presence of 0.5% (w/v) Triton X-100
was taken as 100%. B, size distributions of proteoliposomes
before and after the PEG 3350-mediated fusion as determined by dynamic
light-scattering measurements. Proteoliposomes containing TSecY(Fl51)E
were incubated with (red circles) or without (black
squares) PEG 3350 at 37 °C for 5 min. Liposome sizes were
estimated from light scattering values as described under "Materials
and Methods." C, effects of proteoliposome fusion on the
TSecYE FRET. Proteoliposomes that contained both TSecY(Fl51)E and
TSecY(Rh51)E (molar ratio 1:1) were mixed with those containing a
15-fold excess of unlabeled TSecY(A51C)E. They were then incubated with
(blue) or without (green) PEG for 5 min at
37 °C. Fluorescence spectra were recorded after excitation at 492 nm. The measurements were repeated after detergent (0.5% (w/v) Triton
X-100) solubilization (red and black,
respectively).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Masatada Tamakoshi for helpful suggestions regarding phospholipids from T. thermophilus, Photal Otsuka Electronics for generous support in the dynamic light-scattering experiments, and Kiyoko Mochizuki, Yasuhide Yoshioka, and Michiyo Sano for technical assistance.
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FOOTNOTES |
---|
* This work was supported by Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation (to K. I.), grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to H. M. and K. I.), National Institutes of Health Grant GM26494 (to A. E. J.), and the Robert A. Welch Foundation (to A. E. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB086887 and AB086886.
To whom correspondence should be addressed: Institute
for Virus Research, Kyoto University, Kyoto 606-8507, Japan. Tel.:
81-75-751-4015; Fax: 81-75-771-5699; E-mail:
kito@virus.kyoto-u.ac.jp.
Published, JBC Papers in Press, January 17, 2003, DOI 10.1074/jbc.M300230200
2 Y. Akiyama, unpublished construct.
3 H. Mori, unpublished procedures.
4 Y. Akiyama and K. Ito, manuscript in preparation.
5 S. M. Bhain (2001) Detergents: A Guide to the Properties and Uses of Detergents in Biological System, Calbiochem-Novabiochem Corp., www.calbiochem.com/techresources/RequestLiterature.asp.
6 N. Yamamoto, unpublished results.
7 R. Masui, unpublished information.
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ABBREVIATIONS |
---|
The abbreviations used are: FRET, fluorescence resonance energy transfer; TSecYE, TSecY, TSecE, and TSecA, SecYE, SecY, SecE, and SecA, respectively, from T. thermophilus; C12E8, polyoxyethylene 8 lauryl ether; Ni-NTA, nickel-nitrilotriacetic acid; PEG, polyethylene glycol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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