©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
SecYEG and SecA Are the Stoichiometric Components of Preprotein Translocase (*)

(Received for publication, March 31, 1995; and in revised form, June 12, 1995)

Karen Douville (1) Albert Price (1) Jerry Eichler (1) Anastassios Economou (1) (2)(§) William Wickner (1)(¶)

From the  (1)Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844 and the (2)Department of Biology, University of Crete, 71110 Iraklio, Crete, Greece

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The transport of large preproteins across the Escherichia coli plasma membrane is catalyzed by preprotein translocase, comprised of the peripherally bound SecA subunit and an integrally bound heterotrimeric domain consisting of the SecY, SecE, and SecG subunits. We have now placed the secY, secE, and secG genes under the control of an arabinose-inducible promoter on a multicopy plasmid. Upon induction, all three of the proteins are strongly overexpressed and recovered in the plasma membrane fraction. These membranes show a strong enhancement of 1) translocation ATPase activity, 2) preprotein translocation, 3) capacity for SecA binding, and 4) formation of the membrane-inserted form of SecA. These data establish that SecY, SecE, and SecG constitute the integral membrane domain of preprotein translocase.


INTRODUCTION

Noncytoplasmic proteins are transported into, or across, a membrane as the first stage in their subcellular localization. This process has been studied extensively for endoplasmic reticulum, mitochondria, peroxisomes, chloroplasts, glyoxisomes, and for the plasma membrane of Escherichia coli. The ease of genetic and biochemical approaches in E. coli has made it a model system for such investigations (Schatz and Beckwith, 1990; Bieker et al., 1990; Wickner et al., 1991).

Periplasmic and outer membrane proteins of E. coli are synthesized as preproteins with an amino-terminal leader (signal) sequence (Gierasch, 1989). Some preproteins are stabilized in the cytosol by chaperones such as SecB (Randall and Hardy, 1995). Preprotein-chaperone complexes bind to the plasma membrane at SecA (Hartl et al., 1990), the peripheral membrane subunit of preprotein translocase, driven by the affinities of SecA for SecB (Hartl et al., 1990) and for the leader and mature domains of the preprotein (Lill et al., 1990). SecA itself is bound to the membrane by its affinities for acidic phospholipids (Lill et al., 1990; Hendrick and Wickner, 1991) and for the integral membrane protein SecYEG. The binding of SecA to SecYEG has been shown by the ability of antibody to the SecY amino terminus to reduce SecA binding to membranes (Hartl et al., 1990), the ability of SecA to protect SecY from limited proteolysis (Hartl et al., 1990), and the close agreement between the number of high-affinity binding sites for SecA and the number of SecY molecules in plasma membrane vesicles (Hartl et al., 1990). SecY was functionally isolated as a member of a trimeric complex (Brundage et al., 1990) comprised of SecY, SecE, and SecG (Nishiyama et al., 1994; Douville et al., 1994). This complex is essential for SecA-dependent translocation in proteoliposomes (Brundage et al., 1990). Cross-linking studies have established that the mature domain of a model preprotein lies next to SecA and SecY as it crosses the membrane (Joly and Wickner, 1993).

Translocase uses two sources of energy, ATP (Chen and Tai, 1985) and the protonmotive force Delta (Geller et al., 1986), to drive the preprotein across the membrane. SecA, when associated with the preprotein leader and mature domain, with acidic lipids, and with SecYEG, is activated to utilize ATP in a complex catalytic cycle (Schiebel et al., 1991). In this cycle, a domain of SecA of approximately 30 kDa inserts into, and at least partially across, the membrane, carrying a segment of about 25 aminoacyl residues of the preprotein (Economou and Wickner, 1994). Additional ATP consumption allows this domain to dissociate from the preprotein and de-insert. When the preprotein is not engaged with SecA, further Delta-driven translocation can occur (Schiebel et al. 1991).

Translocation has been reproduced with all-purified components in a reconstituted reaction with proteoliposomes bearing SecYEG, SecA, proOmpA (the precursor of outer membrane protein A), a Delta (established by the incorporation of bacteriorhodopsin in the liposomal membrane) and ATP (Brundage et al., 1990). In this reaction, translocase can support many turnovers of substrate, and the initial rates of translocation per functional translocase complex are similar to those seen with inner membrane vesicles (Bassilana and Wickner, 1993).

Despite extensive biochemical, genetic, and physiological characterization of the translocation reaction, it has remained possible that there are additional subunits of the translocase enzyme. The SecYEG complex is labile (Brundage et al., 1992) and has only been isolated in low yield; other forms of the complex, with additional subunits, might have been missed. Furthermore, genetic and physiological studies have implicated SecD and SecF (Matsuyama et al., 1993; Arkowitz and Wickner, 1994; Pogliano and Beckwith, 1994) and FtsH (Akiyama et al., 1994) in translocation, although the biochemical bases of their actions remain unknown. We now report that inner membrane vesicles prepared from cells with the secY, secE, and secG genes under the ara regulon on a multicopy plasmid are highly enriched in each of the SecYEG subunits. Both catalytic and stoichiometric measurements show a significant increase in translocation activity and sites.


EXPERIMENTAL PROCEDURES

Materials

Inverted inner membrane vesicles (IMVs) were treated with 6 M urea (30 min, 0 °C) to inactivate endogenous SecA, as described by Cunningham et al.(1989). The following proteins were purified according to previous methods: SecA (Cunningham et al., 1989), SecB (Weiss et al., 1988; Lecker et al., 1989), proOmpA (Crooke et al., 1988), and [S]proOmpA (Crooke and Wickner, 1987). NaI (15 mCi/mg) was purchased from Amersham Corp., and [S]methionine (1175 Ci/mmol) was from DuPont NEN. Iodogen (1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril) was from Pierce. Creatine kinase, creatine phosphate, and Proteinase K were from Boehringer Mannheim. Arabinose, ATP, dithiothreitol (DTT), (^1)lipid-free bovine serum albumin (BSA) and L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin were from Sigma. Protein molecular weight markers were from Amersham Corp. (Rainbow markers) and Life Technologies Inc. (^14C-labeled markers).

Plasmid Construction

SecG DNA was amplified by the polymerase chain reaction (PCR) from the Kohara library clone 521 (Kohara et al., 1987; Nishiyama et al., 1993). A clone 521 lysate was diluted 1:10 in sterile water and heated (10 min, 70 °C). A 10-µl aliquot was amplified by PCR using a Coy thermocycler and a GeneAmp kit (Perkin Elmer) following the manufacturer's protocol. Amplification with Amplitaq DNA polymerase was performed for 30 95 °C, 55 °C, and 72 °C cycles. The amino terminus oligonucleotide primer (5`-TAAGGTCATTAAGCTTCTAGAGTATTCACACCCGCTTCAGT) introduced HindIII and XbaI cloning sites, while the carboxyl terminus oligonucleotide primer (5`-GAGATACAGAAAGCTTACAACCATTCCTCAGACCAG) introduced a HindIII cloning site. The PCR product was concentrated by ethanol precipitation and resuspended in sterile water in one-tenth of the original volume. HindIII restriction digests were performed (2 h, 37 °C) on the PCR product and on pHAsecEY DNA (Joly et al., 1994). Each 100-µl digest contained 10 µg of DNA, 50 units, of HindIII (Life Technologies, Inc.), and 10 µl of React 2 (Life Technologies, Inc.). 10 µg of the HindIII-digested pHAsecEY DNA were treated (1 h, 37 °C) with 2 units of shrimp alkaline phosphatase (U. S. Biochemcal Corp.), which were then heat-inactivated (20 min, 65 °C). The phosphatase-treated plasmid DNA and the HindIII-digested PCR product were purified on a 1% SeaPlaque GTG Agarose (FMC) gel in 40 mM Tris acetate, pH 8.0. Ligation was performed by combining 77.3 ng of gel-purified pHAsecEY DNA and 22 ng of gel-purified secG DNA with 1 unit of T4 DNA Ligase (Life Technologies, Inc.) as described previously (Sambrook et al., 1989). The correct orientation of the cloned fragment was confirmed by SmaI-XbaI restriction digestion. pHAsecEYG was transformed into E. coli strain BL21 (Grodberg and Dunn, 1988) as described previously (Ausubel et al., 1994).

Bacterial Growth

All media and antibiotics were according to Sambrook et al.(1989). Cultures were grown at 37 °C with aeration in M9 medium containing 10 µg/ml thiamine, 100 µg/ml ampicillin, 40 µg/ml of each essential amino acid, and 0.2% (w/v) glucose. Exponential cultures at A = 0.5 were induced with 1% (w/v) arabinose and growth continued for 1-3 h. For analysis of total cell proteins, 1 ml of culture was added to 0.5 ml of 30% (w/v) ice-cold trichloroacetic acid (30 min, 0 °C) and centrifuged (10,000 rpm, 4 min, 4 °C). Pellets were twice suspended in 1 ml of ice-cold acetone, vortexed and then centrifuged (14,000 rpm, 10 min, 4 °C). The pellets were air dried (10 min, 37 °C) and resuspended in 50 µl of 1% (w/v) SDS, 1 mM DTT, 20 mM Tris-Cl, pH 8.0 (10 min, 37 °C, with occasional vortexing). Insoluble material was removed by centrifugation (14,000 rpm, 10 min, 23 °C), and 40 µl of the supernatant was transferred to a clean tube and stored at 4 °C.

Membrane Purification

IMVs were prepared by a modification of the method of Chang et al.(1978). Cells from a 5-liter culture were harvested by centrifugation (10,000 g, 5 min, 20 °C), resuspended in an equal weight of 10% (w/v) sucrose, 50 mM Tris-Cl, pH 7.5, frozen dropwise in liquid nitrogen, and stored at -70 °C. Cells were thawed by the addition to 250 ml of room temperature AH buffer (5 mM MgSO(4), 1 mM DTT, 50 mM Hepes-KOH, pH 7.5) and centrifuged (13,000 rpm, 15 min, 4 °C). Pellets were resuspended in 17 ml of AH buffer containing 0.1 mM Pefabloc (Boehringer Mannheim), passed 3 times through a French pressure cell at 8000 p.s.i., and centrifuged (15,000 rpm, 20 min, 4 °C). Supernatants were layered onto 15 ml of 20% (w/v) sucrose in AH buffer and centrifuged in a SW 28 rotor (2 h, 24,000 rpm, 4 °C). Pellets were resuspended in 4 ml of AH buffer with a Dounce homogenizer and then layered onto sucrose step gradients prepared from 6 ml each of 1.6, 1.4, 1.2, 1.0, and 0.8 M sucrose in AH buffer and centrifuged in a SW 28 rotor (16 h, 24,000 rpm, 4 °C). The brown band in the lower third of the gradient was collected and mixed 1:1 with AH buffer and then centrifuged in a 60 Ti rotor (1.5 h, 55,000 rpm, 4 °C). Pellets were resuspended in 0.5 ml of AH buffer and stored at -70 °C.

Iodination of SecA and Assay of Its Membrane Binding and Insertion

SecA was iodinated using Iodogen (Markwell and Fox, 1978) and then used in translocation/membrane insertion reactions (Economou and Wickner, 1994). Binding of I-SecA to urea-treated IMVs (200 µg/ml) prepared from E. coli strain BL21/pHAsecYEG grown with or without arabinose was performed in 50-µl reactions and analyzed according to Scatchard(1949), as described previously (Hartl et al., 1990; Economou and Wickner, 1994). Binding parameters, initially estimated by visual inspection of Scatchard plots (Scatchard, 1949), were determined using the nonlinear, least squares estimates modelling program LIGAND (Munson and Rodbard, 1980; Haberland et al., 1989). The starting estimates were from Hartl et al.(1990). The K (=1/K) value of 23-39 nM and the concentration of SecA receptors (180 pmol/mg of membrane protein) in the membranes of BL21/pHAsecEYG grown in the absence of arabinose are in good agreement with values from Hartl et al.(1990) using KM-9 membranes (K = 30-50 nM and 120-160 pmol of SecA receptor/mg of membrane protein).

Other Methods

Protein concentration was determined using Bradford reagent (Bio-Rad) with BSA as a standard. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (15% polyacrylamide or high Tris gels), fluorography, and electrophoretic transfer of proteins to polyvinylidine difluoride membranes were as described previously (Ito et al., 1980; Douville et al., 1994). Autoradiography of I-labeled polypeptides was performed at -80 °C with intensifying screens.


RESULTS

Regulated Overexpression

The genes encoding an epitope-tagged SecE and wild-type SecY were introduced into a pBR322-derived plasmid under control of the ara regulon (Joly et al., 1994). Oligonucleotide primers were designed to allow PCR amplification of a fragment of DNA bearing the secG gene, using the Kohara library phage 521 as a template. This DNA fragment was then ligated into the plasmid downstream from the secE and secY genes to form plasmid pHAsecEYG, as described under ``Experimental Procedures,'' and transformed into E. coli strain BL21. Arabinose-induced expression resulted in a large increase in the synthesis of proteins of the correct apparent molecular weights for SecY, SecE, and SecG polypeptides (Fig. 1A, lane1). These identifications were confirmed by immunoblot analysis (Fig. 1B). Overexpression of SecY, SecE, and SecG did not induce the synthesis of SecA (Fig. 1B). Pulse-chase analysis showed that each of the three subunits was largely stable after synthesis for 5 min (Fig. 1A, lane 2) or 90 min (data not shown). This confirms earlier studies (Matsuyama et al., 1990) that had shown that SecE overexpression can contribute to the stability of newly synthesized SecY, which is otherwise labile when overexpressed. Upon separation of cell lysates into low speed pellet, high speed supernatant, and inner and outer membrane fractions, it was apparent that the inner membrane fraction was enriched in SecY, SecE, and SecG polypeptides, easily identified in stained gels as major polypeptide constituents (Fig. 1C). Quantitative immunoblot analysis showed a 32-fold overexpression of SecY and a 16-fold overexpression of SecG polypeptides in the overexpressing cells as compared with noninduced controls.


Figure 1: Induction of synthesis of SecY, SecE, and SecG. A, S-pulse label. E. coli BL21 transformed with pHAsecEYG were grown in two 5-ml cultures containing M9 medium, 10 µg/ml thiamine, 100 µg/ml ampicillin, 40 µg/ml of each essential amino acid (except methionine), 0.4% glycerol, and 0.4% maltose to an A of 0.5. One culture was induced by the addition of 0.25 ml of 20% arabinose and grown for 1 h at 37 °C. Cultures were then pulse labeled (1 min, 37 °C) with 400 µCi of [S]methionine protein labeling mix and mixed with an excess of nonradioactive methionine (1 mM). Samples (1 ml) were removed and trichloroacetic acid-precipitated at 0 and 5 min following the onset of the chase. Polypeptides (100,000 cpm/lane) were analyzed by high Tris SDS-polyacrylamide gel electrophoresis (Douville et al., 1994) and autoradiographed for 14 days. Lanes1 and 2, arabinose-induced cells chased for 0 and 5 min, respectively; lanes3 and 4, uninduced cells chased for 0 and 5 min, respectively. B, immunoblot of total cell protein. E. coli BL21/pHAsecEYG was grown, induced with 1% arabinose (2.5 h), precipitated with trichloroacetic acid, and analyzed by high Tris SDS-polyacrylamide gel electrophoresis. Proteins were transferred electrophoretically to polyvinylidine difluoride membranes and probed with antibodies as described previously (Douville et al., 1994). Uninduced (lane1) and induced (lane2) samples were probed with affinity purified alpha-SecY and alpha-SecG antibodies (both at 0.2 µg/ml). Uninduced samples (lane3) and samples induced for expression (lane4) were probed with a mouse monoclonal antibody directed against the hemagglutinin (HA) epitope of HA-tagged SecE, and alpha-SecA antibodies. C, Coomassie Blue-stained gel of IMV polypeptides. E. coli strain BL21/pHAsecEYG was grown as above. Half of the culture was induced with 1% arabinose for 3 h. Both cultures were then used to prepare IMVs. IMV polypeptides were analyzed by high Tris SDS-polyacrylamide gel electrophoresis. Lane1, induced IMV (50 µg/lane); lane2, uninduced IMV (50 µg/lane); and lane3, low molecular weight markers.



Functional Analysis

In the absence of a membrane potential, translocation of preproteins such as proOmpA is driven exclusively by the energy of ATP hydrolysis. Under such conditions, translocation is an inefficient process, resulting in the hydrolysis of thousands of ATP molecules per molecule of translocated proOmpA (Lill et al., 1989). This high level of translocation ATPase is only seen with fully functional translocation sites (Lill et al., 1989; Bassilana et al., 1992). Membranes from wild-type cells (data not shown) or from uninduced BL21/pHAsecEYG cells showed comparable translocation ATPase activity, which was dependent on preprotein, SecA, and membrane vesicles. However, membranes from cells overexpressing the SecY, SecE, and SecG polypeptides, referred to as induced membranes, showed 20 times the translocation ATPase activity as compared with membranes from uninduced cells (Fig. 2). These data suggest that the overproduced SecY, SecE, and SecG are capable of productive interaction with SecA and preproteins and that no other membrane component is limiting for that interaction.


Figure 2: Overproduction of SecY, SecE, and SecG leads to enhanced translocation ATPase activity. Translocation ATPase was measured as described by Lill et al.(1990) with minor modifications. Urea-treated IMVs (100 µg/ml), prepared from either arabinose-induced or uninduced cells as described under ``Experimental Procedures,'' were incubated with SecA (40 µg/ml) and proOmpA (60 µg/ml) in 50 µl at 37 °C for either 2.5 min (membranes from induced cells) or 20 min (membranes from uninduced cells). In control incubations, TL buffer (50 mM KCl, 5 mM MgCl(2), 50 mM Tris-Cl, pH 8.0) containing 10% glycerol or urea buffer (6 M urea, 1 mM DTT, 50 mM Tris-Cl, pH 7.9) was used in place of SecA and proOmpA, respectively. After incubation, 10-µl aliquots were added to 800 µl of malachite green/molybdate solution (Lanzetta et al., 1978) containing 0.1% Triton X-100. After 5 min, 100 µl of 34% citric acid was added. After 40 min at 23 °C, the absorbance at 660 nm was measured. Each point represents the average of 4-6 determinations ± S.D.



To directly examine the functionality of the ``induced membranes,'' translocation reactions were performed with varying chemical concentrations of proOmpA, using purified SecB chaperone, SecA, ATP, and inner membrane vesicles from either induced or uninduced cells. The rate of translocation (expressed as pmol of proOmpA translocated/min/mg of membrane protein) during the initial incubation (when translocation was linear with respect to time) increased at least 30-fold (Fig. 3B), again suggesting that no other membrane component apart from SecYEG is limiting for translocation.


Figure 3: Overexpression of SecY, SecE, and SecG in E. coli BL21 increases the rate of proOmpA translocation into inverted inner membrane vesicles. [S]proOmpA (150,000 cpm), premixed with unlabeled proOmpA, was added to IMVs (100 µg/ml membrane protein), 0.385 µM SecA, 2.2 µM SecB, 2 mM DTT, 2 mM ATP, 10 µg/ml creatine kinase, 5 mM creatine phosphate, 0.5 mg/ml BSA, 5 mM MgCl(2), 50 mM KCl, and 50 mM HEPES, pH 8.0. Delta was imposed by the addition of 2 mM succinate. Reactions were incubated at 37 °C for 10 min (uninduced membranes) or 2 min (induced membranes). Translocation of [S]proOmpA was analyzed by SDS-polyacrylamide gel electrophoresis and fluorography (A) and quantitated by densitometry (B) as described previously (Economou and Wickner, 1994).



Stoichiometric Measures of Translocase

One measure of functionality of the integral domain of translocase is the formation of high affinity sites for SecA binding. Scatchard analysis revealed that the induced membranes had 6 times the number of binding sites (1000-1100 versus approximately 180 pmol of SecA/mg of IMV protein), although the binding affinities of SecA for each membrane were comparable (22 and 37 nM for uninduced and induced membranes, respectively) (Fig. 4). We recently reported that SecA, bound at its high affinity sites, could undergo a reaction of reversible membrane insertion, rendering a domain of approximately 30 kDa inaccessible to added protease (Economou and Wickner, 1994). Membranes from induced cells supported the formation of approximately 8 times the membrane-inserted form of SecA as found with membranes from uninduced cells (Fig. 5). At high concentrations of added SecA, there was clearly saturation of the formation of inserted SecA for membranes from either induced or uninduced cells. These data therefore provide a stoichiometric measure of insertion sites and show that a significant fraction of the overproduced SecYEG subunits are in an active state.


Figure 4: SecA binding to membrane vesicles containing high levels of the SecY, SecE, and SecG polypeptides. The binding of I-SecA to urea-treated IMVs was assayed as described previously (Hartl et al., 1990; Economou and Wickner, 1994). Nonradioactive SecA (0-1396 nM) was mixed with iodinated SecA (218,000 cpm/µg; 4 nM protomer) in TL buffer prior to the addition of IMVs (200 µg of membrane protein/ml). After a 15-min incubation on ice, samples were layered over 5 ml of buffer S (TL buffer containing 0.2 M sucrose, 0.4 mg/ml BSA) and sedimented (35 min, 4 °C, Beckman TLX Optima ultracentrifuge, TLA100 rotor, 70,000 rpm). Radioactivity was measured in the membrane pellet and in the supernatant. For Scatchard binding analysis (Scatchard, 1949), the computer modelling program LIGAND (Munson and Rodbard, 1980) was used.




Figure 5: Membrane insertion of SecA. Protection of the 30-kDa domain of I-SecA from protease was determined as described previously (Economou and Wickner, 1994). Translocation reactions (50 µl) containing I-SecA (80,000 cpm; 0.0456 µg), TL buffer, 200 µg/ml BSA, 30 µg/ml proOmpA, 48 µg/ml SecB, an ATP-regenerating system (5 mM phosphocreatine and 10 µg/ml creatine kinase) and IMVs (100 µg/ml) were prepared on ice. Unlabeled SecA (2-50 µg/ml) in 5 ml of TL buffer containing 10% glycerol was added prior to the addition of membranes. A control sample, marked 0, received 50 µl of SecA buffer alone. The tubes were warmed to 37 °C, and reactions of SecA membrane insertion and proOmpA translocation were initiated with 2 mM ATP. After 15 min, the samples were chilled on ice and digested with 1 mg/ml trypsin (0 °C, 15 min). The membrane-inserted domain of SecA was detected as a protease-protected 30-kDa species by SDS-polyacrylamide gel electrophoresis and autoradiography and quantitated by scanning densitometry (Economou and Wickner, 1994).



To determine whether the overproduced subunits exist as a complex, membranes extracts were prepared with a mixed micellar solution of detergents and phospholipids and chromatographed on an anion-exchange resin. The overproduced SecY, SecE, and SecG subunits co-chromatographed at a position coincident with the translocation ATPase activity of the reconstituted proteoliposomes (Fig. 6), as previously reported for the SecYEG complex from wild-type E. coli (Brundage et al., 1990). This provides direct support for the concept that a considerable portion of the three overexpressed polypeptides are present in the membrane as the active trimeric complex SecYEG.


Figure 6: Purification of overexpressed SecYEG and reconstitution into proteoliposomes. A, purification of SecYEG. IMVs (6 mg) were solubilized in 10 ml of solubilization buffer (1.5% beta-octyl glucoside, 20% glycerol, 3.75 mg/ml E. coli phospholipids, 1 mM dithiothreitol, 20 mM Tris-Cl, pH 7.9) on ice for 90 min. The mixture was centrifuged (24 min, 60,000 rpm, 70.1-Ti rotor) and the supernatant was applied to a DE-52 column (Whatman) (h = 3 cm, d = 0.7 cm), equilibrated in buffer A (Brundage et al., 1990). After elution with 50 ml of buffer A, a 28-ml linear gradient of 0-100 mM KCl in buffer A was applied. 1-ml fractions were eluted into Microfuge tubes containing 500 µl of glycerol, 50 µl of beta-octyl glucoside (12.5% w/v), and 15 µl of E. coli phospholipids (50 mg/ml) and mixed. Aliquots of 40 µl were examined by high Tris SDS-polyacrylamide gel electrophoresis and visualized by silver staining (Blum et al., 1987). B, reconstitution into proteoliposomes. DE-52-purified SecYEG was reconstituted into proteoliposomes by combining 100-µl aliquots with 7 µl of E. coli phospholipids (50 µg/ml), 4 ml of reconstitution buffer (2.5 mM EGTA, 50 mM KCl, 50 mM Tris-Cl, pH 7.9), and 40 µl of CaCl(2) (1 M) overnight on ice. Samples were then centrifuged (20 min, 15,000 rpm, JA-21 rotor), and the pellets were resuspended in 200 µl of TL buffer. Proteoliposomes (30 µl) were examined for translocation ATPase activity as described in the legend to Fig. 2.




DISCUSSION

The integral membrane domain of preprotein translocase has been conserved throughout evolution. SecY and SecE homologs are found in several bacterial species (Ito, 1992; Rensing and Maier, 1994; Murphy and Beckwith, 1994), in yeast (Stirling et al., 1992), in chloroplasts (Scaramuzzi et al., 1992), and in the mammalian endoplasmic reticulum (Görlich and Rapoport, 1993). The mammalian endoplasmic reticulum Sec61 complex has recently been isolated (Görlich and Rapoport, 1993); two of its subunits are homologous to SecY and SecE (Görlich et al., 1992b; Hartmann et al., 1994), while its third subunit is hydrophobic and of similar size to SecG. SecYEG is unusual among permeases in that it transports largely unfolded, membrane-spanning linear aminoacyl polymers with side chains of varying size and charge while maintaining a proton-tight (or, for endoplasmic reticulum, calcium-tight) junction. It is thus important to be certain that all of the relevant subunits of translocase have been defined and to have an abundant supply of this protein for structural characterization. These goals have been addressed in the current study.

Our results, taken together with those from previous studies (Brundage et al., 1990; Hartl et al., 1990; Joly and Wickner, 1993; Joly et al., 1994; Hanada et al., 1994), indicate that SecY, SecE, SecG, and SecA are stoichiometric components of preprotein translocase. It is important, however, to emphasize that additional factors, which are not stoichiometric components of translocase, may play essential catalytic roles. These proteins include leader peptidase, which is essential for the release of translocated preproteins (Dalbey and Wickner, 1985) and (in mammals) the signal recognition particle receptor (Tajima et al., 1986). Other examples of proteins involved in the translocation reaction whose catalytic function is not yet known include SecD and SecF (Pogliano and Beckwith, 1994), FtsH (Akiyama et al., 1994), and (in mammals) TRAM (Görlich et al., 1992a).

How do SecYEG and its eukaryotic homolog, the Sec61 complex, function? Sec61 complex is believed to function as the direct receptor for ribosomes (Kalies et al., 1994), ``gated'' in this function by signal recognition particle and its receptor (Rapoport, 1992) and by the nascent polypeptide associated complex (Wiedmann et al., 1994). No mammalian homolog of SecA, the fourth essential subunit of bacterial transalocase (Oliver, 1993), has yet been described, either structurally or functionally. How then is metabolic energy coupled to Sec61 complex-mediated translocation? ATP may still provide energy via Hsp70 to drive translocation into the endoplasmic reticulum. Lumenal Hsp70 in the endoplasmic reticulum of yeast has a specific binding site on the Sec61 complex and couples ATP hydrolytic energy to preprotein translocation and to Hsp70 release from this site (Brodsky and Schekman, 1993). In some studies, mammalian endoplasmic reticulum translocation also requires ATP (Schlenstedt and Zimmermann, 1987), although lumenal Hsp70 has not been shown to function in the reconstituted system (Görlich and Rapoport, 1993).

In contrast to protein translocation through the Sec61 complex of the mammalian endoplasmic reticulum, most bacterial translocation through SecYEG only begins after a nascent chain critical molecular mass of about 25 kDa (Randall, 1983) is achieved, allowing recognition of the mature domain by SecB (Randall and Hardy, 1994) and SecA (Lill et al., 1990). E. coli translocation may begin after a chain reaches this critical molecular weight but is largely post-translational (Randall, 1983). It is driven by both ATP and Delta in different phases of the translocase catalytic cycle (Schiebel et al., 1991). In the ATP-coupled mode of bacterial translocation, a large 30-kDa domain of SecA penetrates into, and across, the membrane concomitant with translocation of a preprotein segment (Economou and Wickner, 1994). All three SecYEG subunits are required for this SecA-receptor function; overproduction of SecY, SecE, and SecG leads to enhanced SecA binding (Fig. 5), which was not seen with overproduction of SecY and SecE alone (Kim and Oliver, 1994).

SecYEG is essential for the SecA membrane cycling reaction ( Fig. 5and Economou and Wickner(1994)). SecYEG may provide surfaces with which SecA can associate as it undergoes ATP-driven membrane penetration and conformational changes. This might explain why preproteins are found near both SecA and SecY throughout their membrane transit (Joly et al., 1994). SecYEG and SecA may provide a pathway for polypeptide chain movement. The availability of large amounts of SecYEG complex should now facilitate the investigation of these and other structural and catalytic properties of preprotein translocase.


FOOTNOTES

*
This work was supported in part by grants from the National Institutes of General Medical Sciences and the Human Frontier Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by fellowships from UNESCO, EMBO, and HFSPO.

To whom correspondence should be addressed. Tel.: 603-650-1701; Fax: 603-650-1353.

(^1)
The abbreviations used are: DTT, dithiothreitol; BSA bovine serum albumin; Delta, protonmotive force; PCR, polymerase chain reaction; IMVs, inverted inner membrane vesicles; proOmpA, the precursor of outer membrane protein A.


ACKNOWLEDGEMENTS

We thank Marilyn Rice Leonard, Bernadette Hils, and Barbara Atherton for expert assistance and Jon Beckwith and Luz Maria Guzman for the pBAD22 plasmid.


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