Fluorescence Resonance Energy Transfer Analysis of Protein Translocase

SecYE FROM THERMUS THERMOPHILUS HB8 FORMS A CONSTITUTIVE OLIGOMER IN MEMBRANES*

Hiroyuki MoriDagger , Tomoya TsukazakiDagger , Ryoji Masui§, Seiki Kuramitsu§, Shigeyuki Yokoyama, Arthur E. Johnson||, Yoshiaki Kimura**, Yoshinori AkiyamaDagger , and Koreaki ItoDagger DaggerDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta ftsH sfhC21 derivative (13) of strain CU141 (14). Strains JM109 and AD16 (15) were used for DNA manipulation experiments.

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 lacZalpha 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.

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-beta -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-beta -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.

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-beta -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.

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 -80°C.

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 (epsilon 492 = 7.5 × 104 M-1 cm-1; Molecular Probes) or tetramethylrhodamine-5-iodoacetamide (epsilon 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).

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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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%.


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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.

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.


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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 beta -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.

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 beta -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.

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).


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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.

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.


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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.

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.


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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.

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.


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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).

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.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    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.

    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.

Dagger Dagger 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.

    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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