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J Biol Chem, Vol. 275, Issue 4, 2472-2478, January 28, 2000
From the Department of Microbiology and the Groningen Biomolecular
Sciences and Biotechnology Institute, University of Groningen,
Kerklaan 30, 9751 NN Haren, The Netherlands
To determine the phospholipid requirement of the
preprotein translocase in vitro, the Escherichia
coli SecYEG complex was purified in a delipidated form using
the detergent dodecyl maltoside. SecYEG was reconstituted into
liposomes composed of defined synthetic phospholipids, and
proteoliposomes were analyzed for their preprotein translocation and
SecA translocation ATPase activity. The activity strictly required the
presence of anionic phospholipids, whereas the non-bilayer lipid
phosphatidylethanolamine was found stimulatory. The latter effect could
also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer
conformation. Phosphatidylethanolamine derivatives that prefer the
bilayer state were unable to stimulate translocation. In the absence of
SecG, activity was reduced, but the phospholipid requirement was
unaltered. Remarkably, non-bilayer lipids were found essential for the
activity of the Bacillus subtilis SecYEG complex. Optimal
activity required a mixture of anionic and non-bilayer lipids at
concentrations that correspond to concentrations found in the natural membrane.
Complementary genetic and biochemical approaches have shown that
the translocation of proteins across the inner membrane of Escherichia coli is mediated by the translocase (for reviews
see Refs. 1-3). The essential subunits of the translocase are the dissociable peripheral ATPase termed SecA and integral membrane proteins SecY and SecE (4, 5). The SecYE complex forms a preprotein-conducting channel that may associate with SecG or the
heterotrimeric SecDFYajC complex (6). SecA binds with high affinity to
the SecYEG complex and functions as a molecular motor that utilizes the
binding and hydrolysis of ATP to drive the stepwise translocation of a
preprotein across the membrane (7). In addition, the proton motive
force accelerates the translocation reaction (8).
Phospholipids have been shown to play an important role in protein
translocation. The E. coli inner membrane mostly consists of
the zwitterionic phospholipid phosphatidylethanolamine
(PE,1 70-75%) and two
anionic phospholipids, phosphatidylglycerol (PG, 20-25%) and
cardiolipin (CL, 5-10%). The membrane lipid composition is normally
tightly regulated, but by the use of strains engineered in the
expression of critical phospholipid biosynthetic enzymes, the in
vivo manipulation of the bulk phospholipid composition has been
achieved (9). The major anionic phospholipids PG and CL can be depleted
in a strain in which the expression of the pgsA gene
encoding the phosphatidylglycerophosphate synthase is controlled. At
low PG and CL content (2-3%), protein translocation is severely
compromised, whereas lack of only CL did not affect translocation (10).
Only the negative charge of the polar lipid head group is important as
many other anionic phospholipids are able to restore the protein
translocation activity of PG and CL-depleted inner membrane vesicles
(IMVs) or in reconstituted proteoliposomes (4, 11, 12). The efficiency
of protein translocation is directly proportional to the amount of
anionic phospholipids (13). Anionic phospholipids influence various
steps in the preprotein translocation cascade as follows. (i) They
promote the interaction of SecA with the membrane surface (14-16) and
SecYEG (11) and are needed for the SecA translocation ATPase activity,
i.e. the preprotein-stimulated ATPase activity of the
SecYEG-bound SecA (12, 14). At low levels of anionic phospholipids,
excess SecA can compensate for the reduced translocation activity (17), suggesting a role of these lipids in the targeting of preproteins and
SecA to the membrane. In addition, the endogenous SecA ATPase activity
at low Mg2+ concentration is stimulated by the presence of
anionic phospholipids, an activity termed SecA lipid ATPase (14). (ii)
Membrane insertion of the positively charged signal sequence of a
preprotein is dependent on anionic phospholipids (18-22). (iii)
Anionic phospholipids stabilize the SecYEG complex during octyl
glucoside solubilization (12, 23) and (iv) influence the acquisition of
the correct topology of membrane proteins (24).
The cold-sensitive growth defect of a secG null strain
(25-28) can be suppressed by various gene products involved in
phospholipid biosynthesis along with the pgsA gene.
Likewise, the cold-sensitive growth defect of the secAcsR11
mutant strain is suppressed by overproduction of the PgsA protein (28).
These effects have been attributed to an increase in the anionic
phospholipid content that restores the secretion defect of these
strains by facilitating the SecA catalytic cycle at low temperature
(28). The exact mechanism of this activation, however, remains obscure
as studies with photoreactive phospholipid analogues suggest that the
SecYEG-bound, membrane-inserted form of SecA is not in contact with
phospholipids (29, 30).
The other main phospholipid of the E. coli inner membrane is
the type II lipid PE. PE adopts a non-bilayer hexagonal II phase conformation (31). Deletion of the pssA gene (32), which
encodes the phosphatidylserine (PS) synthase, renders cells devoid of the amino-based phospholipids PS and PE. This results in severe pleiotropic effects on membrane protein function and the inactivation of protein translocation (33-35). For growth, this strain is dependent on the presence of a high concentration of divalent cations
(Ca2+, Mg2+, or Sr2+) (32, 35).
These cations stimulate the bilayer to non-bilayer transition of CL,
which comprises 44% of the total phospholipid in this strain (36). It
therefore appears that the polymorphic behavior of these lipids is
essential for growth, a role normally fulfilled by PE. The requirement
for non-bilayer lipids for protein translocation appears less strict
than for anionic lipids (35), but the mechanism by which these lipids
act on protein translocation is unknown. Non-bilayer lipids only
marginally affect the SecA lipid ATPase activity (37) and appear not
essential for the functional reconstitution of protein translocation
using octyl glucoside (OG)-purified SecYEG complex (11).
The reconstitution of preprotein translocation with only a limited set
of purified Sec proteins provides a unique opportunity to assess
systematically the lipid requirement for preprotein translocation. We
now report on a method that allows purification of the SecYEG complex
in a delipidated state. The activity of the delipidated SecYEG can be
restored after reconstitution into liposomes with a defined
phospholipid composition. The data with the purified E. coli
SecYEG complex not only confirm the hypothesis that anionic lipids are
essential for activity but also show that non-bilayer lipids stimulate
the activity of the reconstituted translocase. Strikingly, with the
Bacillus subtilis SecYEG both lipid classes are essential
for activity. Overall, optimal activity is observed when anionic and
non-bilayer lipids are present at a concentration that matches that of
the natural membrane.
Plasmids--
pET610 (38) was partially digested with
NcoI/BamHI, and the large secYE
fragment was cloned into pET302 (39) to yield pET320 that allows the
overproduction of the E. coli SecYE.
Materials--
E. coli SecA (40), B. subtilis SecA (41), SecB (42), pro-OmpA (43), and pre-PhoB (44)
were purified as described. ProOmpA and pre-PhoB were labeled by
iodination with K125I (44) and stored frozen in 6 M urea, 50 mM Tris-Cl, pH 7.5, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
N-methyl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (methyl-DOPE),
N,N-dimethyl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (dimethyl-DOPE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dimyristoyl-oleoyl-sn-glycero-3-phosphoethanolamine (DMPE),
1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]
(DOPS), 1,2 dioleoyl-sn-glycero-3-phosphoglycerol
(DOPG), and dioleoylglycerol (DOG) were from Avanti Polar lipids, Inc.,
Birmingham, AL. Polyclonal antibodies raised against purified
His-tagged SecY and SecE and against a synthetic peptide corresponding
to a SecG domain were obtained as described previously (39).
Purification of Delipidated SecYEG and SecYE Complexes--
IMVs
overexpressing His-tagged SecYE(G) were isolated from E. coli SF100 cells transformed with pET610 (E. coli
SecYEG) (39) or pET320 (E. coli SecYE) (this study) as
described (39). IMVs (60 mg of protein) were solubilized on ice for 30 min at 1 mg/ml in 10 mM Tris-Cl, pH 8.0, and 20% glycerol
(buffer A) supplemented with 2% (w/v) dodecyl maltoside (DDM).
Non-solubilized proteins and aggregates were removed by centrifugation
(30 min at 40,000 × g at 4 °C), and the supernatant
was loaded onto a DEAE column (volume 60 ml) (Whatman, DE52)
equilibrated with buffer A supplemented with 0.03% (w/v) DDM. The
column was washed with 2 volumes of equilibration buffer, and proteins
were eluted with a linear gradient of 0-300 mM KCl in 3 volumes of the same buffer. Alternatively, the SecYEG complex was
extracted from the IMVs and purified with 1.25% (w/v) OG in the
presence or absence of 0.2 mg/ml of E. coli phospholipid.
Fractions were analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue. Purified protein was either stored on ice or frozen in liquid nitrogen and then stored at Reconstitution of SecYEG and SecYE Complexes--
Synthetic
phospholipids were mixed in desired ratios in chloroform solution,
dried under vacuum, washed with ethanol, and dried again. Next, the
lipid film was slowly (1-2 h) hydrated at a final concentration of 10 mg/ml by incubation at room temperature under a nitrogen atmosphere in
a buffer containing 10 mM Tris-Cl, pH 8.0, and 1 mM dithiothreitol. The suspension was finally dispersed by
the use of a bath sonicator. For reconstitution, 100 µl of purified
E. coli SecYEG or SecYE protein (about 0.1 mg/ml) was mixed
with 20 µl of lipids (10 mg/ml), incubated for 5 min on ice, and
diluted rapidly with 8 ml of 50 mM Tris-Cl, pH 8.0, 50 mM KCl. After 5 min, proteoliposomes were collected by
centrifugation (30 min, Ti-70, 200,000 × g at 4 °C)
and resuspended in 100 µl of 50 mM Tris-Cl, pH 8.0, and
50 mM KCl. Proteoliposomes were analyzed for the amount of
incorporated protein using the DC Protein assay (Bio-Rad), by 15%
SDS-PAGE stained with Coomassie Brilliant Blue, and by immunoblotting
on polyvinylidene difluoride membranes using antibodies directed again
SecY, SecE, and SecG. Reconstituted SecYE(G) proteoliposomes were
frozen and stored in liquid nitrogen. Before use, samples were thawed
at 37 °C and sonicated 3 times for 10 s in a bath sonicator.
Control experiments showed that the SecA translocation ATPase activity
after rapid dilution was linear with the amount of SecYEG added at
lipid-to-protein ratios (w/w) above 10.
To reconstitute the B. subtilis SecYEG into liposomes of
different lipid compositions, IMVs were isolated from E. coli SF100 cells transformed with pET822 that directs the
functional overexpression of the B. subtilis SecYEG complex
(45). IMVs (100 µl; 0.1 mg of protein/ml) were solubilized in 1.25%
(w/v) OG in buffer A as described (39). After 30 min incubation on ice,
the non-solubilized material was removed by centrifugation (TLA100.4,
30 min, 180,000 × g at 4 °C). The supernatant
fraction (100 µl) was subsequently mixed with 20 µl of lipids (10 mg/ml), rapidly diluted, and isolated as described above. In control
experiments, IMVs were used derived from E. coli SF100 cells
transformed with pET610 that overproduce E. coli SecYEG
(38).
Fusion of SecYEG Proteoliposomes--
SecYEG (proteo)liposomes
(100 µl; containing 0, 10, or 20 µg of SecYEG protein and 0.2 mg of
phospholipids) were mixed with an equal volume and amount of
(proteo)liposomes of different lipid composition. Samples were quickly
frozen in liquid nitrogen and slowly thawed on ice. This procedure was
repeated three times. Before use in activity assays, samples were
sonicated 3 times for 10 s in a bath sonicator.
Translocation Assays--
Translocation assays were performed in
a final volume of 50 µl consisting of 50 mM HEPES-KOH, pH
7.6, 30 mM KCl, 5 mM Mg(Ac)2, 2 mM ATP, 10 mM creatine phosphate, 0.5 µg of
creatine kinase, 25 µg of bovine serum albumin, 1.6 µg of SecB, 1 µg of purified E. coli or B. subtilis SecA, and
proteoliposomes containing 6.5 µg of SecYEG or SecYE complex. Samples
were preincubated for 10 min at 37 °C, and the translocation
reaction was started by the addition of 1 µl of
125I-labeled pro-OmpA or pre-PhoB (0.2 mg/ml in 6 M urea, 50 mM Tris-Cl, pH 7.5). After 10 min,
reactions were terminated by chilling on ice. Samples were treated with
proteinase K (0.1 mg/ml) for 15 min, precipitated with ice-cold 10%
(w/v) trichloroacetic acid, acetone-washed, and analyzed by SDS-PAGE on
10 (pre-PhoB) and 12% (pro-OmpA) polyacrylamide gels.
Other Analytical Techniques--
Translocation ATPase activity
of urea-treated IMVs or SecYEG proteoliposomes was measured with
pro-OmpA as substrate as described (46). Protein concentration was
determined with the DC Protein assay (Bio-Rad). Phospholipid phosphorus
was assayed after heat destruction of chloroform/methanol-extracted
phospholipids using the method of Rouser et al. (47).
Purification and Delipidation of SecYEG Complex--
For the
purification of the OG-solubilized SecYEG complex, the presence of at
least 0.2 mg/ml E. coli phospholipids is essential to retain
activity (5, 12, 23). To assess the phospholipid dependence of the
translocase in a systematic manner, it is necessary to deplete the
purified SecYEG complex of endogenous lipids. When OG was replaced with
DDM, the addition of phospholipids was no longer required to purify the
SecYEG complex in a functional state. DDM-solubilized and -purified
SecYEG complex was as active as complex purified from OG-solubilized
membranes in the presence of added phospholipids (Table
I). The final amount of endogenous phospholipid present in the DDM-solubilized SecYEG complex was below
the detection limit, which corresponds to less than 3 mol of
phospholipid per mol of SecYEG complex, assuming an equimolar stoichiometry of the subunits. In contrast, the OG-purified enzyme retained about 200 mol of phospholipid per mol of SecYEG. These data
demonstrate that the SecYEG complex can be purified in a delipidated
and functional state with the detergent DDM.
Reconstitution of SecYEG and Lipid Dependence--
Delipidated
SecYEG complex was used to examine the phospholipid requirement of
protein translocation. The phospholipid composition of the E. coli inner membrane corresponds to about 70 mol % of the
non-bilayer lipid PE and 30 mol % of the acidic PG and CL. To mimic
the native polar head group composition, the SecYEG complex was
reconstituted into liposomes composed of 70 mol % of DOPE and 30 mol
% of DOPG. The amount of DOPC was gradually replaced by DOPG to
analyze the requirement for anionic phospholipids. Total amounts of
SecY, SecE, and SecG reconstituted in the liposomes were determined by
Western blotting and were equal for each of the proteoliposomes (data
not shown). Proteoliposomes were supplemented with SecA and analyzed
for the SecA ATPase activity in the absence and presence of the
precursor pro-OmpA (Fig. 1B)
and for the translocation of chemical amounts of
125I-labeled pro-OmpA (Fig. 1A). The activity
was determined after 10 min of incubation, and within this time
interval, the amount of translocated pro-OmpA increased linearly in
time (data not shown). SecYEG was completely inactive when
reconstituted into DOPC:DOPE (30:70, molar ratio) (Fig. 1, A
and B) but became activated when the DOPC was replaced for
DOPG or DOPS (Fig. 1A). A similar observation was made for
the endogenous and pro-OmpA-stimulated SecA ATPase activity (Fig.
1B). The requirement for the other main lipid constituent of
the E. coli inner membrane, PE, was determined by
reconstitution of the SecYEG complex into liposomes composed of
DOPG:DOPC (30:70, molar ratio) whereby the DOPC was gradually replaced
for DOPE. Unlike DOPG, DOPE appears not to be essential for protein
translocation activity. A low but significant translocation (Fig.
1C) and translocation ATPase (Fig. 1D) activity was observed with the SecYEG complex reconstituted in the absence of
DOPE. However, DOPE markedly stimulated the activity of SecYEG to
3-fold. These results show that anionic phospholipids are essential for
the reconstitution of a functional translocase using purified and
delipidated SecYEG, whereas PE is stimulatory.
Phospholipids Reversibly Influence Translocase Activity--
To
exclude the possibility that the observed phospholipid requirement is
due to a difference in reconstitution efficiency, the SecYEG complex
was first reconstituted into proteoliposomes composed of a lipid
mixture that does not support activity. Subsequently, other lipids were
introduced into these inactive proteoliposomes by freeze-thawed induced
fusion with liposomes of different phospholipid composition. SecYEG
proteoliposomes composed of DOPC were essentially inactive (2%
activity) for the pro-OmpA-stimulated SecA ATPase activity (Fig.
2). Activity could, however, be restored
by fusion of these proteoliposomes with DOPG:DOPC:DOPE (30:20:50, molar ratio) liposomes yielding a final lipid composition of DOPG:DOPC:DOPE (15:60:25, molar ratio). The activity of these fused proteoliposomes was about 65% of a control in which the SecYEG complex was
reconstituted directly into this lipid mixture (Fig. 2). In another
experiment, SecYEG was reconstituted with DOPG:DOPC (30:70, molar
ratio) yielding proteoliposomes that support a low SecA translocation
ATPase activity. Re-introduction of DOPE into these proteoliposomes by
fusion with DOPC:DOPE (50:50, molar ratio) liposomes, again yielding a
final lipid composition of DOPG:DOPC:DOPE (15:60:25, molar ratio),
resulted in an increase of the activity to about 60% of the control.
These results demonstrate that the activity of the reconstituted SecYEG complex can be reversibly modulated by the bulk phospholipid
composition.
Non-bilayer Lipids Stimulate Protein Translocation--
To examine
further the effect of the non-bilayer lipid PE on protein
translocation, the head group and acyl chain properties were varied. In
a lipid mixture of DOPC:DOPG (70:30, molar ratio), the DOPC was
gradually replaced for either DOPE, methyl-DOPE, or dimethyl-DOPE.
Although introduction of DOPE significantly stimulated the pro-OmpA
translocation activity (Fig.
3A) and SecA translocation
ATPase (Fig. 3B) of SecYEG proteoliposomes, this effect was
largely abolished when the DOPE was mono- or dimethylated. When the
DOPE was exchanged for dimyristoyl-PE (DMPE), a PE derivative with a
shorter hydrophobic acyl chain, hardly any stimulation of the activity
was observed (Fig. 3). Since the methylated forms of PE and DMPE are
all bilayer lipids (31, 48), it appears that the stimulatory effect of
DOPE is indeed due to its ability to form non-bilayer structures. To
confirm this hypothesis further, the effect of dioleoylglycerol (DOG),
a lipid with strong non-bilayer forming properties, was examined. DOG
markedly stimulated the activity of the SecYEG complex to the same
extent as DOPE (Fig. 3). Taken together these data demonstrate that PE
affects protein translocation by its ability to adopt a non-bilayer
conformation.
The Lipid Requirement of the SecYE Complex Is Not Affected by
SecG--
To determine if the presence of SecG influences the lipid
requirement of translocation, the SecYE complex was purified from an
overexpressing strain and reconstituted into various lipid mixtures.
Although some endogenous SecG co-purified with SecYE, Western blotting
demonstrated that the amount of SecG in the SecYE proteoliposomes was
at least 25-fold lower than in the SecYEG proteoliposomes (data not
shown). The absence of SecG resulted in a dramatic reduction of the
activity of the SecYE complex. However, the requirement for anionic
phospholipids (DOPG) and non-bilayer lipids (DOPE) (Fig.
4) as observed with the SecYE complex was
indistinguishable from that found for SecYEG. This indicates that there
is no direct mechanistic relation between the SecG function and the
activating effect of lipids.
Lipid Requirement of the B. subtilis SecYEG Complex--
To
determine if the lipid requirement found for the E. coli
SecYEG complex extends to other bacterial species, the lipid
specificity of the B. subtilis SecYEG complex was
determined. Unlike E. coli, anionic phospholipids are the
major constituents of the B. subtilis cytoplasmic membrane,
i.e. 70% PG, 4% CL, and 12% PE (49). To obtain functional
B. subtilis translocase, the SecY, SecE, and SecG proteins
were overproduced in E. coli (45). IMVs derived from cells
overexpressing either the B. subtilis or E. coli
SecYEG were solubilized in OG without the addition of exogenous lipids and directly reconstituted into a 20-fold excess of synthetic lipids by
rapid dilution. A mixture of DOPG:DOPE (70:30, molar ratio) was used to
mimic the lipid composition of B. subtilis. Either the DOPG
or DOPE was replaced for DOPC to analyze the requirement for anionic
and non-bilayer lipids, respectively. The translocation activity of the
B. subtilis SecYEG complex was assayed in the presence
of purified B. subtilis SecA and 125I-labeled
pre-PhoB, a Bacillus-specific precursor (44, 45). 125I-Pro-OmpA was used with the E. coli
SecYEG proteoliposomes. Translocation of pro-OmpA by the
E. coli SecYEG proteoliposomes again showed the strict
requirement for PG and stimulation by PE (Fig.
5). The presence of 25 mol % of PG and
30 mol % of PE in the E. coli SecYEG proteoliposomes was
already sufficient to saturate the activity (see also Fig. 1,
C and D). Therefore, it seems that the E. coli SecYEG is most active in a synthetic lipid mixture that
resembles the polar head group composition of the E. coli inner membrane. Remarkably, translocation of pre-PhoB by B. subtilis SecYEG proteoliposomes appeared much more critical with
respect to the lipid composition. Maximal translocation activity
required a very high DOPG concentration (Fig. 5) and was optimal in a
lipid mixture corresponding closely to the polar head group composition of the B. subtilis membrane, i.e. DOPG:DOPE
(70:30, molar ratio) (Fig. 5). The data suggest that the requirement
for anionic and non-bilayer lipids for preprotein translocation is a
general feature of bacterial translocase complexes and further show
that in B. subtilis non-bilayer lipids are essential for
activity.
The E. coli preprotein translocase depends for its
activity on specific classes of phospholipids. Previous in
vivo and in vitro studies have shown that anionic
phospholipids are essential for activity, whereas non-bilayer lipids
are stimulatory (for review see Ref. 51). A systematic study of the
phospholipid requirement of the purified translocase has not been
reported despite the fact that the lipid composition of the
reconstituted proteoliposomes can be manipulated in a convenient and
systematic manner. We now show that functional reconstitution of the
purified, delipidated E. coli SecYEG requires anionic
phospholipids, whereas non-bilayer lipids stimulate translocation.
These results confirm the studies performed in the crude membrane
system but, in addition, extend these observations to the B. subtilis SecYEG. Remarkably, the activity of the B. subtilis SecYEG strictly depends on the presence of non-bilayer
lipids. For optimal activity, anionic and non-bilayer lipids need to
present at amounts corresponding to the phospholipid composition of the
native inner membrane.
In order to analyze the phospholipid requirement of the purified
translocase, it is desirable to first delipidate the enzyme and
subsequently restore its activity by reconstitution in liposomes with a
defined phospholipid composition. The SecYEG complex has been
purified from IMVs after solubilization with OG. However, to obtain a
functional SecYEG complex it is necessary to include phospholipids in
the buffers used during the purification (Refs. 5, 12, and 23 and this
study). Delipidation of OG-purified SecYEG leads to the irreversible
inactivation of the enzyme (Table I). We now show that SecYEG can be
purified in a delipidated and functional form when DDM is used as a
detergent instead of OG. OG is a detergent with a rather short acyl
chain of only eight carbon moieties. The acyl chain of DDM is longer
and thus may more closely resemble the interaction of the phospholipid
acyl chain with the SecYEG complex present in detergent micelles. In this respect, it was previously noted that OG-solubilized SecYEG is
thermolabile (39, 51, 52). DDM-purified SecYEG complex is less
susceptible to such thermal
inactivation.2 The
DDM-purified SecYEG complex, like the OG-purified, lipid-supplemented SecYEG complex (39), supports the high affinity binding of SecA, the
nucleotide-induced conformational states of SecA, and the endogenous
SecA ATPase activity.3
However, it has not been possible to detect SecA translocation ATPase
activity with the detergent-solubilized translocase even though the
enzyme is stable at the temperatures that support this activity in
reconstituted liposomes. Electron microscopic studies on the B. subtilis SecYE complex indicate the presence of oligomeric forms
that may constitute the preprotein conducting channel (53). This
oligomeric form may be a stable state of the SecYE complex, but
alternatively, the SecYE oligomer may be a dynamic entity, disassembling and assembling in response to the demand for
translocation. When reconstituted into a lipid membrane, the kinetics
of such an assembly event will largely be dictated by the lateral
protein diffusion rate. In detergent micelles, however, assembly will be limited by the rate of collision within the three-dimensional space
and/or the ability of the micelles to fuse. These events may be
effective only when the enzyme is present at a very high concentration.
Strikingly, preprotein translocation in detergent solution has been
reported for the Sec61p isolated from the endoplasmic membrane of yeast
(54) but is observed only at a very high Sec61p concentration.
Although the purification method in the absence of phospholipids was
developed to be able to examine the effects of small quantities of
phospholipids on the activity of the translocase, the data show that
high concentrations of anionic and non-bilayer lipids are needed to
saturate the translocation activity. It thus appears that phospholipids
act on protein translocation in a more global sense. The E. coli SecYEG complex is maximally active in a mixture of 30% DOPG
and 70% DOPE. As far as the ratio between anionic and non-bilayer
lipids concerns, this mixture "more or less" corresponds to the
lipid composition of the E. coli inner membrane. The
activity in the optimal synthetic mixture is about 75% of that found
with natural E. coli phospholipids, showing that substantial
activity can be recovered with the defined system. Reconstituted
B. subtilis SecYEG complex is maximally active in 70% DOPG
and 30% DOPE, i.e. at a ratio that closely matches that of
anionic to non-bilayer lipids in the native B. subtilis
inner membrane. It is remarkable that both systems differ in their
quantitative lipid requirement and are most active at their
physiological lipid conditions. In this respect, the lipid requirement
of the E. coli SecYEG complex did not change significantly
when pre-PhoB translocation was assayed3 instead of
pro-OmpA.
Anionic phospholipids have been shown to fulfill at least a dual role,
i.e. they promote SecA membrane binding and insertion and
stimulate the interaction of the signal sequence of preproteins and the
membrane (50). These lipids may indirectly stimulate targeting of SecA
to the SecYEG complex, for instance by promoting the low affinity
membrane binding of SecA. This may result in a membrane-bound pool of
SecA protein that could have a kinetic advantage relative to the
cytosolic pool to associate with SecYEG complexes that have completed a
translocation reaction. Experimental evidence for such mechanism is,
however, difficult to obtain. Another possible role for anionic
phospholipids may be found in the putative assembly of the SecYEG
complex into larger functional oligomers. The collective cold-sensitive
secretion defect of the secG null and secAcsR11
strain (28) and many other Sec mutants may be found in a compromised
channel assembly activity.
The reconstitution studies with the purified SecYEG complex provide
compelling evidence that non-bilayer lipids stimulate translocation.
The activating effect of DOPE can be mimicked with dioleoylglycerol, a
lipid that like DOPE adopts a non-bilayer conformation, whereas
bilayer-forming PE derivatives fail to stimulate translocation. These
data provide strong evidence that the lipid shape is the major factor
for activation rather than the amino group of PE. In contrast to the
E. coli SecYEG, the activity of the B. subtilis
SecYEG strictly required the presence of non-bilayer lipids. In
contrast, the amino group of PE is needed for the functional reconstitution of the lactose permease of E. coli (55, 56) and leucine permease of Lactococcus lactis (57). PE
stimulates the folding of the lactose permease into its active
conformation (58, 59). Likewise, non-bilayer lipids may be needed for
folding and/or assembly of the SecYEG complex.
Our data suggest that the absolute amounts of bilayer (or anionic) and
non-bilayer lipids are essential for optimal activity. Since these
lipids are needed at high concentration, it seems that the protein
translocation activity is determined by the collective, physical
properties of the membrane. The requirement for non-bilayer lipids
could relate to their effect on the lateral membrane pressure and/or
optimal matching of the protein-lipid interface and thus affect the
conformation of the active translocase.
We thank Jeanine de Keyzer, Erik Manting,
Gert Moll, and Andreas Veenendaal for technical assistance and fruitful discussions.
*
This work was supported by a PIONIER grant of the
Netherlands Organization for Scientific Research (N.W.O.) and by CEC
Biotech Grants BIO2 CT 930254 and BIO4 CT 960097.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.
2
A. Veenendaal, manuscript in preparation.
3
C. van der Does, J. Swaving, W. van Klompenburg,
and A. J. M. Driessen, unpublished results.
The abbreviations used are:
PE, phosphatidylethanolamine;
PG, phosphatidylglycerol;
PS, phosphatidylserine;
CL, cardiolipin;
DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine;
methyl-DOPE, N-methyl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;
dimethyl-DOPE, N,N-dimethyl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;
DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;
DMPE, 1,2-dimyristoyl-oleoyl-sn-glycero-3-phosphoethanolamine;
DOPS, 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine];
DOPG, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol;
DOG, dioleoylglycerol;
OG, n-octyl
Non-bilayer Lipids Stimulate the Activity of the Reconstituted
Bacterial Protein Translocase*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Purification of the E. coli SecYEG complex
View larger version (36K):
[in a new window]
Fig. 1.
Anionic lipid and phosphatidylethanolamine
requirement for the functional reconstitution of the E. coli
SecYEG complex. Dodecyl maltoside-purified SecYEG was
reconstituted in liposomes composed of the indicated synthetic
phospholipids and analyzed for the ATP-dependent
translocation of 125I-pro-OmpA (A/C) and the
SecA ATPase activity in the presence (filled bars) and
absence of pro-OmpA (open bars) (B/D).
View larger version (22K):
[in a new window]
Fig. 2.
Reversible activation of reconstituted SecYEG
by DOPE. SecYEG was reconstituted in proteoliposomes of the
indicated lipid compositions, and as indicated by the
arrows, equal volumes of proteoliposomes were fused by
repeated freeze-thawing steps. The amount of reconstituted SecYEG was
0, 10, and 20 µg, indicated by , +, and ++, respectively. The
pro-OmpA-stimulated SecA ATPase activity of the original and fused
proteoliposomes was measured as described under "Experimental
Procedures." The activity of the SecYEG reconstituted into liposomes
composed of DOPG:DOPC:DOPE (15:60:25, molar ratio) which had undergone
similar freeze thawing cycles was set to 100%.
View larger version (27K):
[in a new window]
Fig. 3.
Non-bilayer lipids stimulate preprotein
translocation. Purified SecYEG complex was reconstituted in
DOPG:DOPC (30:70, molar ratio) proteoliposomes in which the DOPC was
gradually replaced for DOPE (open circles), methyl-DOPE
(open squares), dimethyl DOPE (closed squares),
DMPE (open triangles), or DOG (closed circles).
Proteoliposomes were supplemented with SecA and assayed for the
ATP-dependent translocation of 125I-pro-OmpA
(A) and for the SecA-ATPase activity in the absence and
presence of pro-OmpA (B).
View larger version (29K):
[in a new window]
Fig. 4.
Lipid dependence of SecYEG and
SecYE-mediated pro-OmpA translocation. Purified SecYE
and SecYEG was reconstituted into liposomes with the indicated lipid
compositions and analyzed for the translocation of
125I-labeled pro-OmpA.
View larger version (31K):
[in a new window]
Fig. 5.
Comparison of the lipid dependence of
B. subtilis and E. coli
SecYEG-mediated preprotein translocation. IMVs
containing highly overproduced B. subtilis or E. coli SecYEG complex were solubilized in octyl glucoside and
reconstituted in proteoliposomes with the indicated lipid compositions.
Proteoliposomes were supplemented with purified B. subtilis
or E. coli SecA protein, and analyzed for the
ATP-dependent translocation of 125I-labeled
pre-PhoB or pro-OmpA as indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 31-50-3632164;
Fax: 31-50-3632154; E-mail: A.J.M.DRIESSEN@BIOL.RUG.NL.
ABBREVIATIONS
-glucopyranoside;
DDM, dodecyl maltoside;
IMVs, inner membrane vesicles;
PAGE, polyacrylamide
gel electrophoresis.
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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