(Received for publication, March 31, 1995; and in revised form, June 12, 1995)
From the
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.
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 (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
-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
(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.
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
-SecY and
-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
-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.
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, 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
, 50 mM KCl, and 50 mM HEPES, pH 8.0.
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).
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% -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
-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
(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.
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
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.