©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Effect of Precursor Ribose Binding Protein of Escherichia coli and Its Signal Peptide on the SecA Penetration of Lipid Bilayer (*)

(Received for publication, January 18, 1996)

Taeho Ahn Hyoungman Kim (§)

From the Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Taejon, 305-701, Korea

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Digestion of vesicle-bound SecA by trypsin entrapped within the vesicles showed that refolding precursor ribose-binding protein (pRBP) of Escherichia coli retards the lipid bilayer penetration by SecA while the signal peptide enhances it. This discrepancy was found to be due to reduced SecA binding to the vesicles in the presence of the pRBP while the signal peptide induced a tight binding. Studies on the binding of 1-anilino-8-naphthalene sulfonate (ANS) to SecA indicated that SecA assumes more closed conformation upon interaction with pRBP and signal peptide induces more open structure of SecA. Kinetic studies of ANS binding to SecA upon dilution of unfolded pRBP with SecA solution showed an initial fast ANS binding, which was followed by a slow release of ANS. This suggests that first the signal peptide portion of the pRBP binds with the SecA making its structure more open and then the subsequent binding of the mature domain makes the SecA structure more compact. The pRBP enhanced the digestion of SecA added to the E. coli inverted vesicles, suggesting an inhibition of SecA penetration while the signal peptide had an opposite effect, agreeing with the results from the model systems above. When the pRBP and ATP were present together, however, the penetration of SecA increased dramatically underlining the importance of the SecY/E complex for the membrane insertion of SecA.


INTRODUCTION

Proteins translocated across the plasma membrane of Escherichia coli are synthesized as precursors with amino-terminal signal peptides which contain the information for the membrane targeting. It has been demonstrated that signal peptides interact with many components of the export machineries (Akita et al., 1990; Altman et al., 1990; Bieker et al., 1990). Targeting of various precursor proteins to the plasma membrane of E. coil seems to be carried out through two routes (Wolin, 1994). One subgroup of precursor proteins, such as maltose-binding protein, are post-translationally translocated and initially binds with SecB. Proteins of another subgroup, including the ribose-binding protein (RBP), (^1)are translocated with a targeting mechanism very close to the one used by secretory proteins in mammalian cells. The nascent precursor proteins of the latter subgroup initially bind to the Ffh protein (Phillips and Silhavy, 1992), which has a high amino acid sequence homology with the 54-kDa subunit of the signal recognition particle in mammalian cells (Römisch et al., 1989). These two different targeting routes in E. coli, however, converge at SecA with which the precursor proteins bind. After this stage, the precursor proteins are translocated across the membrane by SecY/E and some additional membrane proteins through an as yet unknown mechanism.

SecA protein, which has a pivotal role among a number of protein components of the translocation machinery, is an ATPase (Lill et al., 1989) and displays some unusual physical properties. In particular, it can exist as a water-soluble form, a peripheral protein, and an integral protein (Cabelli et al., 1991). The basal ATPase activity is stimulated by its interaction with signal peptide and the mature domain of the precursor proteins, negatively charged phospholipids, and SecY/E complex (Lill et al., 1990). Since most of the ATPases involved in the membrane transport or locomotion have ATP/ADP cycle closely linked to their cyclic change in conformation (and also membrane topology for the integral protein ATPases), it has been speculated that the ATP hydrolysis cycle of the SecA is also coupled to its cyclic conformational and topological changes, which are closely linked to the translocation of precursor proteins (Economou and Wickner, 1994; Kim et al., 1994; Ahn and Kim, 1994).

In a preliminary communication (Ahn and Kim, 1994), we reported that the SecA protein traverses the lipid bilayer and its membrane topology depends on the kind of the nucleotide present, indicating the possibility of topology change coupled to the ATP/ADP cycle of the SecA. Here, we extended this study to investigate the effect of the precursor RBP (pRBP) and its signal peptide on the membrane topology of SecA. Since the isolated wild-type signal peptide has a low solubility in water, we used a revertant signal peptide (SF). The first mutation of L(-17)P in the wild-type signal peptide abolishes the translocation, but an additional mutation within the signal peptide of S(-15)F to form the SF peptide largely recovers the translocation capability (Park et al., 1988). The structural studies showed that both wild-type and revertant peptides have a high propensity for alpha-helix formation, but its mutant peptide has a low alpha-helix content (Yi et al., 1994; Chi et al., 1995).


EXPERIMENTAL PROCEDURES

Materials

Cibacron Blue Sepharose, phenyl-Sepharose, and proteinase K were from Pharmacia Biotech Inc. GdnHCl, trypsin, phosphatidylethanolamine (from bovine brain), dioleoyl phosphatidylglycerol, and IODO-GEN were from Sigma. Stock solutions of GdnHCl were prepared daily, and their concentrations were determined by refractometry. Protein concentration was determined by the Bradford method using bovine serum albumin as a standard (Bradford, 1976). [-P]ATP (3000 Ci/mmol) and NaI (2 µl; 1 mCi) were from Amersham Corp.

SecA Preparation

SecA protein was purified from a SecA-overproducing strain (RR1/pMAN400) (Kawasaki et al., 1989). The SecA-enriched fraction after 40-50% ammonium sulfate precipitation was dialyzed against a buffer containing 25 mM Tris-HCl and 1 mM DTT (pH 7.5). This solution was loaded onto a Cibacron Blue Sepharose column. The column was washed with a buffer containing 25 mM Tris-HCl, 1 mM DTT, and 0.3 M KCl (pH 7.5) and then eluted with another buffer containing 25 mM Tris-HCl, 1 mM DTT, and 1.3 M KCl (pH 7.5). Fractions containing SecA were pooled and stored at -75 °C. The purity of this preparation as assayed by SDS-PAGE and densitometry was close to 100%.

Preparation of RBP

pRBP and mature RBP (mRBP) were purified from the strains IQ87 (MC4100 secY/pCI857, pSP107) and SP114 (NR 69/pSP107), respectively, by ion exchange chromatography as described in detail elsewhere (Teschke et al., 1991) but after some minor modifications. The ammonium sulfate precipitation following the sonication of cells was replaced with CM-Sepharose column chromatography.

Preparation of Signal Peptide

The revertant signal peptide SF, in which the Leu at -17 position in the wild-type peptide is replaced by a Pro and Ser at -15 position is replaced by a Phe, was synthesized by a solid phase method on a MilliGen (Burlington, MA) model 9060 automated peptide synthesizer. A second revertant signal peptide, TI, where, in addition to the amino acid replacement at -17 position, the Thr at -18 position was substituted with an Ile, was also synthesized. The peptides were purified by reverse-phase high performance liquid chromatography using a Phenomenex W-porex C(8) column (15 cm times 1.0 cm), elution being made with a water-acetonitrile linear gradient (10-45% of acetonitrile) containing 0.1% trifluoroacetic acid. The sequences of these peptides were confirmed by a MilliGen/Biosearch 6600 Prosequencer.

Digestion of SecA by Vesicle-entrapped Trypsin

Large unilamellar vesicles (LUV) were prepared with phosphatidylethanolamine and dioleoyl phosphatidylglycerol (60:40, by weight) by reverse phase evaporation method (Szoka et al., 1978). The phospholipid concentration was determined using the method of Vaskovsky et al.(1975). Trypsin was encapsulated within the vesicles by the method used by Dumont and Richards(1984) and by Rietveld et al.(1986). Digestion of SecA by vesicle-entrapped trypsin was carried out by mixing solutions of SecA, vesicles, and unfolded pRBP together and then incubating at 30 °C. These components were dissolved in a buffer containing 1 mM DTT, 2 mM MgCl(2), 20 mM HEPES (pH 7.5) and the final concentrations were 3 µM for SecA, 0.6 mM for phospholipid (trypsin encapsulated LUV), and 10 µM for trypsin inhibitor. pRBP was initially unfolded in 1 M GdnHCl but the final GdnHCl concentration after mixing was low enough for the pRBP to have the native structure as shown by circular dichroism and fluorescence spectroscopy. For some experiments, 4.5 µM SF peptide, 6 µM native pRBP, or 6 µM refolding mRBP was replaced the refolding pRBP. Here as well as in the subsequent experiments, the refolding RBP is operationally defined as initially unfolded RBP in 1 M GdnHCl being refolded after dilution with a solution containing SecA. These experiments were performed with or without the presence of ATP or its nonhydrolyzing analog, ATPS. The digestion reactions were terminated by adding SDS sample buffer containing 3 mM phenylmethanesulfonyl fluoride and then placing the reaction vessel in an ice bath. The samples were boiled for 5 min prior to the electrophoresis on a denaturing 12% (w/v) sodium dodecyl sulfonyl polyacrylamide gel (Laemmli, 1970).

Quenching of SecA Trp Fluorescence by Vesicle-entrapped Iodide

KI was entrapped within the vesicles as was described before (Ahn and Kim, 1994). Solutions of SecA, iodide-entrapped vesicles, and unfolded pRBP in 1 M GdnHCl, all in the same buffer solution as in the SecA digestion (pH 7.5), were mixed together and incubated at 30 °C for 30 min before fluorescence measurements. Fluorescence intensity was measured using a Shimazu RF5000 spectrofluorometer in a thermostatted cuvette. The excitation wavelength for the intrinsic tryptophan fluorescence was 295 nm.

SecA Binding to Phospholipid Vesicles

Purified SecA was iodinated using IODO-GEN (Markwell and Fox, 1978). Free iodine was removed by Sephadex G25 chromatography. Binding experiments were performed with 1 µMI-labeled SecA and 0.7 mM phospholipid (LUV) in the presence of either 4 µM refolding pRBP or 2 µM SF signal peptide in a buffer containing 50 mM potassium phosphate, 100 mM KCl, and 1 mM DTT (pH 7.5). Protein-bound vesicles were centrifuged in a Beckman TLA 100.2 rotor at 70,000 rpm for 1 h at 4 °C, and the radioactivities of pellets and supernatant were determined using a Beckman model 5500 counter.For the extractability experiments, the SecA bound to phospholipid vesicles in the presence or absence of SF signal peptide was treated with various concentrations of potassium chloride and urea. Vesicles were sedimented, and the radioactivities of pellets and supernatant were counted as described above.

Conformational Change of SecA

Possible conformational changes in SecA upon interaction with either refolding pRBP or the SF signal peptide under various conditions were monitored by fluorescence spectroscopy. Exposure of Trp of the SecA was tested by mixing SecA with either SF peptide, TI peptide, refolding pRBP, refolding mRBP, or combination of SF and refolding mRBP. All experiments were performed at 30 °C with 0.4-8.0 µM SecA, 4-fold concentration of refolding pRBP, 4-fold concentration of refolding mRBP, or 2-fold concentration of signal peptides in 50 mM potassium phosphate buffer, pH 7.5. pRBP and mRBP were first unfolded in 1 M GdnHCl solution, and the final GdnHCl concentration in the reaction samples after dilution was 0.02-0.04 M in all the experiments. The solutions of SF, TI, and native pRBP also contained 0.02-0.04 M GdnHCl. Fluorescence emission spectra were obtained as before.

For the iodide quenching of Trp fluorescence, a series of KI solutions with the concentration up to 0.1 M were added to the reaction mixtures to obtain the same total concentration of KI plus KCl but different KI concentration.

Changes in the exposure of hydrophobic patches on the SecA upon interaction with either refolding pRBP or the signal peptides were also monitored with fluorescence from 200 µM ANS, which was added to reaction samples. The fluorescence emission caused by excitation at 370 nm was measured between 420 and 600 nm.

Effect of pRBP on the Stability of SecA

Thermal unfolding of SecA was monitored by following the decrease in fluorescence intensity at 338 nm (excitation 295 nm) with a 1 °C/min heating rate.

Kinetic Studies of SecA-pRBP Interaction

In order to check possible sequential interactions of the signal peptide and the mature domain of pRBP with SecA, a solution containing ANS and pRBP unfolded in 1 M GdnHCl was rapidly mixed with a 2 µM SecA solution and the ANS fluorescence intensity at 470 nm was followed. The final concentration of ANS and pRBP was 200 and 8 µM, respectively, and the unfolded pRBP was diluted 50-fold. Control experiments were repeated either without pRBP or after replacing of pRBP with SF signal peptide.

The kinetic progress of conformational change of SecA brought about by refolding pRBP was also studied by time-dependent SecA binding to phenyl-Sepharose. 0.1 µMI-SecA (3 times 10^5 cpm/µg) was incubated with 0.2 µM unfolded pRBP for different time periods at 30 °C, after which the phenyl-Sepharose, suspended in 50 mM potassium phosphate (pH 7.5), was added to the reaction mixtures. After 1 min of incubation, samples were centrifuged for 10 s. Radioactivities of the pellets and supernatants were determined using a Beckman model 5500 counter. From these the percentages of SecA bound to phenyl-Sepharose were estimated.

ATP Binding to SecA

2 µM SecA in 20 mM Tris-HCl (pH 7.3) was incubated with various concentrations of ATP containing 1/1000 as much [-P]ATP. Binding experiments were carried out in the presence of either the signal peptide or the refolding pRBP. After 30 min of incubation at 30 °C, 0.1 volume of each sample was filtered through a spun concentrator with molecular weight cutoff of 10,000. The radioactivity of each filtrate was determined by a liquid scintillation counter (Beckman LS6000LL) and compared with the control values without proteins.

Digestion of SecA Bound to the Outer Surface of IMV

To compare with the digestion of SecA by the entrapped trypsin within the phospholipid vesicles, we performed the proteinase K digestion of SecA bound to E. coli inverted inner membrane vesicle (IMV). IMVs were prepared from E. coli CP626 (MC4100 flhDrbsB102::Tn10; Kim et al., 1992) as described by Chang et al.(1978) and treated with 6 M urea to inactivate SecA (Cunningham et al., 1989). The concentrations of proteins and signal peptide were the same as used in the digestion of SecA by vesicle-entrapped trypsin. Here again, ATP or ATPS was also added for some experiments. 2 mg/ml IMV and 5 µg/ml protease K were incubated at 30 °C for 15 min. The reaction was stopped by the addition of SDS sample buffer containing 3 mM phenylmethanesulfonyl fluoride as in the case of trypsin digestion and followed by SDS-PAGE. Proteins separated on an SDS-polyacrylamide gel were transferred to a nitrocellulose filter. Rabbit anti-SecA serum was diluted 1000-fold and used in conjunction with goat anti-rabbit immunoglobulin G-horseradish peroxidase conjugate as a secondary antibody after 1000-fold dilution.


RESULTS

Effect of the pRBP and the Signal Peptide on the Penetration of SecA into Phospholipid Vesicles

Fig. 1shows the SDS-PAGE of the fragments after 1 h of digestion of external SecA by trypsin entrapped within phospholipid vesicles. This is a sufficient time to reach the steady-state level of digestion (Ahn and Kim, 1994). The digestion was performed in the presence of initially unfolded pRBP, unfolded mRBP, or the SF signal peptide. The effect of ATP or ATPS was also examined. When only the SecA was present (lane a), fragments with molecular masses of 67, 60, 31, and 20 kDa were obtained. The 98-kDa band is the intact SecA and the band near the 20-kDa mark is from trypsin inhibitor. Two minor bands of SecA fragments can also be seen between the intact SecA and 67-kDa bands. There are some minor differences between this pattern and the one shown previously (Ahn and Kim, 1994), which may arise from the difference in phospholipid compositions. It was not possible to determine the topology of the SecA from these multiple fragments.


Figure 1: Effects of pRBP and SF signal peptide on the topology of SecA in the lipid bilayer. SecA was incubated (1 h, 30 °C) with phospholipid vesicles entrapped with trypsin, trypsin inhibitor, and either unfolded pRBP or signal peptide SF. The digestion products were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue.



An appreciable reduction in the density of the intact SecA can be seen when signal peptide SF was present (lane b), indicating an enhanced penetration of SecA. Here, we used a revertant signal peptide in lieu of the wild-type signal peptide because the latter peptide is insoluble in buffer solutions. Compared with the case when SecA alone is present (lane a), there is no change in the band positions but the density of the 30-kDa band increased while those of the other fragments remained about the same. A Met-rich 25-residue peptide of the Ffh protein of E. coli, which showed a higher propensity of forming amphiphilic alpha-helix (^2)like RBP signal peptides (Yi et al., 1994; Chi et al., 1995) had no effect. Addition of either ATP (lane c) or its non-hydrolyzing analog, ATPS (lane d) did not affect the intensity of intact SecA, but there were some major changes in the fragments.

A dramatic reduction in the digestion of SecA was seen when refolding pRBP interacted with SecA (lane e). The thick band around 30 kDa is the pRBP. It is possible that a small amount of 30-kDa fragment of SecA may be also present. In any case, it is clear that unfolded pRBP reduced the lipid bilayer crossing of SecA, unlike the signal peptide, which increased the membrane penetration of SecA. The refolding mRBP (lane h) and folded pRBP (data not shown) had no effect. It is interesting that the refolding mature domain of pRBP overshadowed the effect of the signal peptide, which enhances the bilayer penetration of the SecA. It is likely that the pRBP forms a complex with SecA while it is being folded. The presence of either ATP (lane f) or ATPS (lane g) in addition to refolding pRBP did not affect the digestion pattern (lane e).

The retardation of SecA penetration by refolding pRBP was also demonstrated by the reduction of the fluorescence quenching of the Trp in SecA by iodide entrapped within the vesicles. SecA has seven Trp, but there is no Trp in pRBP. Fig. 2(line a) gives the Trp fluorescence spectrum of SecA bound to vesicles, and line b shows the quenching by vesicle-entrapped iodide. These have also been shown in a previous report (Ahn and Kim, 1994). When refolding pRBP is also present, a tremendous reduction of quenching occurred (line d) even after an allowance for the increase in the fluorescence intensity when pRBP interacts with SecA bound to vesicles without iodide was made (line c). It is clear that the pRBP reduces the SecA crossing of the lipid bilayer, thus lessening the exposure of Trp to the iodide present within the internal space of the vesicles.


Figure 2: Effect of pRBP on the quenching of SecA Trp fluorescence by iodide entrapped within vesicle. 0.4 µM SecA was incubated with 0.5 mM (phospholipids) of vesicles entrapped with iodide for 15 min at 30 °C. 1.2 µM unfolded pRBP was added to this mixture and further incubated for 15 min. Trp fluorescence emission spectra of SecA were obtained with 295 nm excitation wavelength. Ivesicle, phospholipid vesicle entrapped with iodide; vesicle, vesicles without entrapped iodide.



Binding of SecA to Vesicles

Fig. 3shows the binding of SecA to a model membrane with the same lipid composition as in Fig. 1. Here, the SecA sedimented with the vesicles is shown. More than 80% of the native SecA originally added to the solution was bound to the vesicles. Although the presence of the SF peptide appears to increase the SecA binding, this increase is within the estimated experimental error. The addition of native pRBP also did not affect the SecA binding appreciably. A tremendous reduction in the SecA binding was observed when refolding pRBP was present.


Figure 3: Inhibition of SecA binding to liposomes by pRBP. I-labeled SecA was incubated with phospholipid vesicles in the absence or presence of unfolded pRBP and signal peptide. Vesicles with bound SecA was pelleted by ultracentrifugation, and the radioactivities of pellets and supernatant were determined by -ray counting.



Fig. 4shows that the bilayer bound SecA is harder to extract with high concentration of KCl or urea in the presence of the signal peptide. From these binding results, we may conclude that the reduced amount of SecA fragments in the presence of refolding pRBP (Fig. 1) originated from the inhibitory effect of pRBP on the SecA binding to vesicles.


Figure 4: The extractability of membrane-bound SecA protein. Membrane-bound I SecA with or without signal peptide was treated with indicated concentration of KCl or urea and sedimented by ultracentrifugation. The amount of membrane-bound SecA was determined by -ray counting.



The Effect of pRBP and Signal Peptide on the Intrinsic Tryptophan Fluorescence of SecA

The Trp emission spectra of SecA obtained in the presence of refolding pRBP or signal peptide with the excitation at 295 nm are shown in Fig. 5. Refolding pRBP caused a decrease in fluorescence intensity by 12%, and revertant signal peptides increased the intensity by about 30-40% as compared with that of free SecA. However, there was no change in the (max). We did not observe any change in fluorescence intensity when either refolding mRBP or native pRBP was added. This suggests that only refolding pRBP and signal peptides influence the structure of SecA. The final concentration of GdnHCl in these experiment was 0.04 M, and this concentration had no effect on the intrinsic fluorescence intensity of SecA (data not shown). In the presence of 0.04 M GdnHCl, pRBP has the same conformation as that of native pRBP as assayed by tyrosine fluorescence and circular dichroism at 222 nm (data not shown).


Figure 5: Intrinsic tryptophan fluorescence spectra of SecA. All solutions contained 0.2 µM native SecA. Solutions with added pRBP or signal peptide are indicated. SecA was incubated with either 0.8 M pRBP or signal peptide for 1 h at 30 °C prior to the tryptophan fluorescence emission spectra were recorded as described under ``Experimental Procedures.'' The final concentration (0.02 M) of GdnHCl was adjusted to be the same in all reaction samples.



Fig. 6gives quenching of the SecA Trp fluorescence by iodide in the presence of either refolding pRBP or the SF signal peptide. The fluorescence intensity at the emission wavelength of 338 nm was measured. The fluorescence data are plotted according to the Stern-Volmer equation (Eftink and Ghiron, 1981), where F(o) is the emission intensity in the absence of iodide, F is the intensity in the presence of iodide, K is the Stern-Volmer quenching constant, and [I] is the molar concentration of iodide.


Figure 6: Quenching of tryptophan fluorescence of SecA by iodide. 0.5 µM free SecA (open circles), in the presence of 2.0 µM refolding pRBP (filled triangles) or 1.0 µM signal peptide (open squares) was prepared and titrated with KI. F(o) and F were determined as described under ``Experimental Procedures.''



The K value estimated from the slope was 2.55 M for the native SecA. This value increased to 3.18 M in the presence of refolding pRBP and decreased to 1.37 M when the signal peptide was added. It is clear that more SecA Trp residues are exposed to the surface when refolding pRBP is present, but less Trp residues are exposed to the solvent when the signal peptide is present.

The Effect of pRBP and Signal Peptide on the ANS Binding to SecA

Fig. 7shows that the native SecA binds ANS significantly. This suggests that SecA has exposed hydrophobic regions even in the native structure. When the signal peptide SF was present together with SecA, the fluorescence intensity of ANS increased by about 30%. TI peptide increased the ANS fluorescence intensity even more. Since the signal peptides themselves showed no detectable binding of ANS, it is clear that additional hydrophobic sites of SecA are exposed when signal peptides are bound. However, upon addition of pRBP unfolded in 1 M GdnHCl, the hydrophobic sites of SecA were reduced as evidenced by a decrease in ANS fluorescence intensity by about 29% when compared with that of free SecA. The final concentration of GdnHCl after mixing was 0.02 M and under this condition SecA appears to assume the native structure. This conclusion was drawn from the observation that fluorescence intensity of SecA-bound ANS in 0.02 M GdnHCl was the same as the case of no GdnHCl (data not shown). A control experiment showed no ANS binding to pRBP within the GdnHCl concentration of up to 1.5 M. pRBP is fully unfolded in 0.8 M GdnHCl, and the transition midpoint concentration is 0.47-0.48 M when measured by tyrosine fluorescence and circular dichroism (data not shown). This suggests that either partially or fully unfolded pRBP has no hydrophobic clusters to which ANS may bind. pRBP refolding in the presence of ANS also did not show any fluorescence. The reduction in ANS fluorescence intensity by the refolding pRBP suggests that the mature domain of the RBP converts the SecA into more compact form by overcoming an opposing effect by the signal peptide covalently linked to it. mRBP alone had no effect. When equal mole amounts of signal peptide and refolding mRBP were added together to SecA solution, the result was the same as the case when signal peptide alone was added.


Figure 7: Fluorescence emission spectra of ANS bound to SecA. 1 µM SecA alone (solid line), with refolding pRBP (dashed line), or with signal peptide (dotted line) was incubated for 1 h and then ANS was added. Excitation wavelength was 370 nm, and all other conditions were the same as described in Fig. 5.



Sequential Interaction of the Signal Peptide and the Mature Domain of Refolding pRBP with SecA

Fig. 8(line b) shows the time-course of ANS binding to SecA when unfolded pRBP was diluted with SecA solution. This may be compared with line a, which is a control experiment without pRBP. There is an initial sharp increase in ANS binding above the control reaching the level of another control experiment when the SF peptide is present (line c). This is then followed by a gradual decrease below the control value (line a). The time course of ANS binding suggests that the signal peptide first binds with the SecA, making its structure more open, and then the subsequent binding of the mature domain makes the SecA structure even more compact than the native SecA structure.


Figure 8: Kinetic progress curves of ANS binding to SecA as measured by the fluorescence at 470 nm (at 30 °C). Unfolded pRBP or signal peptide and ANS solution were mixed with SecA solution before the measurement was started. Line a, SecA alone; line b, SecA + pRBP; line c, SecA + signal peptide.



The kinetic progress profile of the SecA binding to phenyl-Sepharose in the presence of refolding pRBP qualitatively agrees with the above kinetics of ANS binding to SecA. Upon dilution of unfolded pRBP with SecA solution, about 55% of SecA was bound to phenyl-Sepharose initially within the dead time of mixing (Fig. 9), then SecA was released reaching to 37% after about 40 s. When only SecA was present, about 48% of the total SecA was bound to phenyl-Sepharose which did not change with time. This result indicates that the initial interaction of unfolded pRBP with SecA induced significant increase in the exposure of hydrophobic sites on SecA. But the hydrophobicity of SecA decreased with a time scale similar to that of the release of ANS from SecA (Fig. 8, line b).


Figure 9: Effect of unfolded pRBP on the binding of SecA to phenyl-Sepharose. I-SecA was incubated with unfolded pRBP for the indicated time period, and then phenyl-Sepharose was added. Samples were centrifuged, and radioactivities of pellets and supernatant were determined using a -counter.



pRBP Increases the Stability of SecA

Fig. 10shows the thermal unfolding transition profile of SecA measured by tryptophan fluorescence emission at 338 nm with excitation at 295 nm. Native SecA showed transition temperature, T(m), near 41 °C, which is similar to the result obtained by Ulbrandt et al.(1992). However, the addition of refolding pRBP resulted in a significant increase in the T(m) to 45 °C. This result unequivocally demonstrated that pRBP stabilizes SecA, which apparently is due to the conversion of SecA into a more compact structure.


Figure 10: Thermal transition of SecA. Change in the fluorescence intensity at 338 nm were recorded as a function of temperature. First, unfolded pRBP was refolded by rapid dilution with SecA solution and incubated for 30 min at 30 °C. The solution was then cooled to 17 °C before starting the thermal transition measurements. Excitation was made at 295 nm. The concentrations of SecA and pRBP were 0.2 and 0.8 µM, respectively. T, thermal transition temperature.



Effects of Signal Peptide and pRBP on ATP Binding to SecA

Fig. 11shows the effect of both refolding pRBP and signal peptide on the ATP binding to SecA. When SecA alone is present, the binding approaches the value of 1 mol of ATP/mol of SecA. This is similar to the high affinity ADP binding to SecA (Mitchell and Oliver, 1993). When refolding pRBP is also present, the ATP binding is reduced to about half of the value when SecA alone is present. The situation becomes more complex when the SF signal peptide is present together with SecA. The extent of binding at low ATP concentration is less than the case of no SF peptide, but it becomes greater at higher ATP concentration. Here again, the effect of pRBP on the ATP binding to SecA is just opposite of the signal peptide. Although this should be related to the opposite effects of these on the structure of SecA, the precise nature of the effect is obscure at the moment. It should be noted that the addition of refolding pRBP released ATP bound to SecA. This result agrees with the earlier observation that proOmpA causes the release of nucleotides bound to SecA in free solution (Shinkai et al., 1991). However, these results appear to contradict with the observation that the interaction of the preproteins with the membrane-bound SecA stimulates the ATPase activity of SecA (Lill et al., 1990). On the other hand, the synthetic signal peptides by themselves competitively inhibited the enzyme activity and the denatured mature proteins had no effect (Lill et al., 1990).


Figure 11: ATP binding of SecA. 2 µM SecA in 20 mM Tris-HCl (pH 7.3) was incubated with ATP containing [-P]ATP in the presence of either 4 µM SF signal peptide or 6 µM unfolded pRBP for 30 min at 30 °C. Each sample was filtered through a spun concentrator, and the bound ATP was determined by counting the radioactivity of filtrate.



Digestion of the SecA Bound to IMV by Externally Added Protease

This was performed with IMVs obtained from the strain of E. coli CP626 (rbs102::Tn10). Digestion of the bound SecA by external proteinase K at 30 °C produced major proteolytic fragments with molecular mass of about 75 and 67 kDa (Fig. 12). Addition of either ATP or refolding pRBP greatly increased the digestion, indicating less SecA penetration into IMV membrane. On the other hand, the presence of SF signal peptide retarded the digestion, suggesting that this peptide increased the SecA penetration. These results are consistent with the results obtained with the model membrane (Fig. 1).


Figure 12: Immunoblot analysis of protease K digests of SecA. SecA was mixed with IMVs prepared from E. coli CP626. pRBP unfolded in 1 M GdnHCl or signal peptide was added to this solution and incubated for 20 min at 30 °C. Samples were treated with proteinase K for 15 min at 30 °C and analyzed by SDS-PAGE, followed by immunoblotting with anti-SecA antiserum. The positions of molecular weight markers and SecA (98 kDa) are indicated by arrows. The control is obtained by immunoblotting of the SDS-PAGE gel of the IMV without the presence of added protein. The total protein concentration (w/v) was adjusted to be the same in all samples by adding the appropriate amount of carbonic anhydrase.



When refolding pRBP and ATP were added together, however, the digestion pattern was reversed as shown in Fig. 13. When only SecA was present with IMV and proteinase K was added, a faint band of 67 kDa plus the intact SecA band could be seen. When these were mixed with unfolded pRBP and ATP, the bands became more dense and three additional bands with smaller molecular weight were also produced. Apparently, the combined effect of pRBP and ATP brought about an increase in SecA penetration into IMV membrane. It should also be noticed that all these bands disappeared after a long period (more than 20 min) of digestion, demonstrating the SecA penetration into IMV membrane is a reversible process.


Figure 13: Proteinase K digestion of IMV-bound SecA in the presence of ATP and refolding pRBP. ATP and unfolded pRBP was added to the solution containing IMV-bound SecA and proteinase K digestion was performed for the indicated time period at 30 °C. The rest of the procedures were the same as described in Fig. 12.




DISCUSSION

The importance of interaction of SecA with anionic phospholipids of E. coli for translocation of preprotein as well as in vivo ATPase activities of SecA has already been shown (Lill et al., 1990; de Vrije et al., 1988; Kusters et al., 1991). The extent of in vivo SecA binding to inner membrane of E. coli was found to be dependent upon the presence of anionic phospholipids (Lill et al., 1990). SecA interaction with model membrane of acidic phospholipid was indirectly demonstrated by an increased SecA ATPase activity (Lill et al., 1990) and by the susceptibility of SecA to staphylococcal protease V8 (Shinkai et al., 1991) when liposome was added to SecA solution. Breukink et al. (1992) also showed that the penetration of SecA into monolayer containing negatively charged lipids is influenced by binding and hydrolysis of ATP.

The actual demonstration of SecA penetration into lipid bilayer was observed by Ulbrandt et al.(1992) using fluorescence quenching as a means of determining penetration depths. A deep penetration at least the depth of outer monolayer was demonstrated, but it was not possible to decide whether SecA traverses the bilayer. They also observed a partial unfolding of SecA accompanying the penetration. Our previous study established that SecA protein traverses the lipid bilayer completely and that the penetration is inhibited by ATP while ADP promotes it (Ahn and Kim, 1994). This conclusion was drawn from the digestion of externally added SecA by the trypsin entrapped within the phospholipid vesicles. The digestion products were analyzed by SDS-PAGE, and the electrophoresis pattern was found to be dependent on the kind of nucleotide present. The present investigation shows that the isolated signal peptide enhances the digestion of SecA by vesicle-entrapped trypsin while refolding pRBP has an opposite effect. Refolding mRBP with or without the presence of isolated signal peptide did not influence the SecA digestion. It was shown that SecA binds more extensively to the vesicles in the presence of signal peptide and less in the presence of refolding pRBP as compared to the binding when SecA alone is present.

The reason why the signal peptide promotes the binding of SecA to the vesicles and subsequent penetration of bilayer, while the refolding pRBP has an opposite effect, seems to be that signal peptide converts the SecA into a more open form while refolding pRBP makes it more compact. This conclusion was drawn from the following experimental observations. First, more ANS bind to SecA in the presence of SF peptide than in the absence of the peptide, while refolding pRBP has an opposite effect (Fig. 7). It is clear that, in the presence of SF signal peptide, more hydrophobic patches of SecA were exposed to the surface, which increased ANS binding. Second, the presence of refolding pRBP increased the thermal stability of SecA (Fig. 10), and this protein becomes more resistant to the denaturation by GdnHCl. In view of the stabilizing effect of refolding pRBP on SecA structure, it is somewhat surprising that refolding pRBP decreased the intrinsic Trp fluorescence intensity, which indicates that Trp residues are more extensively exposed to the solvent in the presence of refolding pRBP. There is no easy explanation for this, but this appears to be a common phenomenon judging from the reports for the ATP-induced conformational change of the hsp90 and DnaK (Csermely et al., 1993; Palleros et al., 1992), among others. Although this differential mode of interaction of signal peptide and refolding pRBP with SecA explains the effect of these on the SecA penetration of lipid bilayer, the reason for the difference is not entirely clear. It is possible that the initial structural change brought about by the signal peptide binding may facilitate the subsequent binding of the mature domain, which converts the SecA into more compact form. This was borne out by the kinetics of structural change in SecA when this interacts with refolding pRBP ( Fig. 7and Fig. 8). Since the refolding mRBP by itself or mRBP together with the isolated signal peptide had no effect, it is clear that this occurs only when the mature domain is covalently linked to the signal peptide. The essence of the results obtained here is that, when refolding pRBP interacts with SecA, signal peptide is the one first binds with SecA making it more lipid bilayer penetrable, and this paves the way for the binding of the mature domain, which, in turn, converts the SecA into membrane-inaccessible form. This is an interesting observation by itself regardless of its relevance to the in vivo translocation of pRBP and merits further investigation to elucidate the underlining mechanism.

As to the relevance of the observation we made here to the pRBP translocation, the results with the IMV are of interest. It is noteworthy that ATP and refolding pRBP separately affect the SecA penetration into IMV just as they did to SecA insertion into lipid bilayer. However, when ATP and refolding pRBP were mixed with IMV at the same time, an opposite effect of more extensive SecA penetration can be seen. This confirms the results by Kim et al.(1994), who used proOmpA at 0 °C using the immunoblotting with anti-SecA antiserum as was here. Although the results obtained by Economou and Wickner(1994) are somewhat different from our results as well as from those by Kim et al.(1994), possibly because of different band identification method, the general conclusion that SecA penetrates the IMV membrane in the presence of preprotein and ATP is the same. Another interesting aspect of our result is that the SecA and its fragments disappear very rapidly (Fig. 13). The time-dependent change in the band density was not investigated either by Kim et al.(1994) or by Economou and Wickner(1994). But the latter authors demonstrated the reversible nature of SecA penetration from exchange between inserted and deinserted forms during translocation. This indicates the dynamic nature of the topologies of intact SecA as well as its fragments. It is possible that, under the condition given, SecA and its fragments oscillate between insertion and deinsertion modes and they are accessible to proteinase K during the deinsertion phase. Only an extensive kinetic studies will be able to shed light on this.

The experimental results with IMV also underline the importance of the integral protein members of the translocation machinery, especially the SecY/E complex. It was proposed that the preprotein translocation is carried out by protein-conducting channels, which are opened by signal peptides (Simon and Blobel, 1992). From the cross-linking experiments, it was also suggested that only SecA and SecY are in contact with precursor proteins during translocation (Joly and Wickner, 1993), suggesting a possibility that the channel forming proteins are SecY and SecA. Any model of protein-conducting channel involving SecA must accommodate the insertion-deinsertion cycle of this protein. It is possible that equilibrium as well as kinetic studies of SecA penetration into vesicles reconstituted with SecY/E in the presence of refolding pRBP and ATP may be needed to clarify: 1) the difference in this individual and combined effects of pRBP and ATP, and 2) how SecA can be a part of a channel and still act as a ``shuttle.''


FOOTNOTES

*
This study was supported in part by the Korea Science and Engineering Foundation. 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.

§
To whom correspondence should be addressed. Tel.: 82-42-869-2602; Fax: 82-42-869-2610.

(^1)
The abbreviations used are: RBP, ribose-binding protein; pRBP, precursor RBP; mRBP, mature RBP; IMVs, inverted inner membrane vesicles; GdnHCl, guanidine hydrochloride; ANS, 1-anilino-8-naphthalene sulfonate; LUV, large unilamellar vesicle; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; ATPS, adenosine 5`-O-(thiotriphosphate).

(^2)
D.-B. Oh and H. Kim, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Myeong-Jun Choi of Mogam Biotechnology Research Institute for the synthesis of the signal peptide.


REFERENCES

  1. Ahn, T., and Kim, H. (1994) Biochem. Biophys. Res. Commun. 203, 326-330 [CrossRef][Medline] [Order article via Infotrieve]
  2. Akita, M., Sasaki, S., Matsuyama, S., and Mizushima, S. (1990) J. Biol. Chem. 265, 8164-8169 [Abstract/Free Full Text]
  3. Altman, E., Bankaitis, V. A., and Emr, S. D. (1990) J. Biol. Chem. 265, 18148-18153 [Abstract/Free Full Text]
  4. Bieker, K. L., Phillips, G. J., and Silhavy, T. (1990) J. Bioenerg. Biomembr. 22, 291-310 [Medline] [Order article via Infotrieve]
  5. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  6. Breukink, E., Demel, R. A., de Korte-Kool, G., and de Kruijff, B. (1992) Biochemistry 31, 1119-1124 [Medline] [Order article via Infotrieve]
  7. Cabelli, R. J., Dolan, K. M., Qian, L., and Oliver, D. B. (1991) J. Biol. Chem. 266, 24420-24427 [Abstract/Free Full Text]
  8. Chang, C. N., Blobel, G., and Model, P. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 361-365 [Abstract]
  9. Chi, S.-W., Yi, G.-S., Suh, J.-Y., Choi, B.-S., and Kim, H. (1995) Biophys. J. 69, 2703-2709 [Abstract]
  10. Csermely, P., Kajtar, J., Hollosi, M., Jalsovszky, G., Holly, S., Kahn, C. R., Gergely, P., Jr., Söti, C., Mihaly, K., and Somogyi, J. (1993) J. Biol. Chem. 268, 1901-1907 [Abstract/Free Full Text]
  11. Cunningham, K., Lill, R., Crooke, E., Rice, M., Moore, K., and Wickner, W. (1989) EMBO J. 8, 955-959 [Abstract]
  12. de Vrije, T., de Swart, R. L., Dowhan, W., Tomassen, J., and de Kruijff, B. (1988) Nature 334, 173-175 [CrossRef][Medline] [Order article via Infotrieve]
  13. Dumont, M., and Richards, F., M. (1984) J. Biol. Chem. 259, 4147-4156 [Abstract/Free Full Text]
  14. Economou, A., and Wickner, W. (1994) Cell 78, 835-843 [Medline] [Order article via Infotrieve]
  15. Eftink, M. R., and Ghiron, C. A. (1981) Anal. Biochem. 114, 199-227 [Medline] [Order article via Infotrieve]
  16. Joly, J. C., and Wickner, W. (1993) EMBO J. 12, 255-263 [Abstract]
  17. Kawasaki, H., Matsuyama, S., Sasaki, S., Akita, M., and Mizushima, S. (1989) FEBS Lett. 242, 431-434 [CrossRef][Medline] [Order article via Infotrieve]
  18. Kim, J., Lee, Y., Kim, C., and Park, C. (1992) J. Bacteriol. 174, 5219-5227 [Abstract]
  19. Kim, Y. J., Rajapandi, T., and Oliver, D. B. (1994) Cell 78, 845-853 [Medline] [Order article via Infotrieve]
  20. Kusters, R., Dowhan, W., and de Kruijff, B. (1991) J. Biol. Chem. 266, 8659-8662 [Abstract/Free Full Text]
  21. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  22. Lill, R., Cunningham, K., Brundage, L. A., Ito, K., Oliver, D., and Wickner, W. (1989) EMBO J. 8, 961-966 [Abstract]
  23. Lill, R., Dowhan, W., and Wickner, W. (1990) Cell 60, 271-280 [Medline] [Order article via Infotrieve]
  24. Markwell, M. A. K., and Fox, C. F. (1978) Biochemistry 17, 4807-4817 [Medline] [Order article via Infotrieve]
  25. Mitchell, C., and Oliver, D. B. (1993) Mol. Microbiol. 10, 483-497 [Medline] [Order article via Infotrieve]
  26. Palleros, D. R., Reid, K. L., McCarty, J. S., Walker, G. C., and Fink, A. L. (1992) J. Biol. Chem. 267, 5279-5285 [Abstract/Free Full Text]
  27. Park, S., Liu, G., Topping, T. B., Cover, W. H., and Randall, L. L. (1988) Science 239, 1033-1035 [Medline] [Order article via Infotrieve]
  28. Phillips, G. J., and Silhavy, T. J. (1992) Nature 359, 744-746 [CrossRef][Medline] [Order article via Infotrieve]
  29. Randall, L. L. (1985) EMBO J. 4, 1875-1880 [Abstract]
  30. Rietveld, A., Jordi, W., and de Kruijff, B. (1986) J. Biol. Chem. 261, 3846-3856 [Abstract/Free Full Text]
  31. Römisch, K., Webb, J., Herz, J., Prehn, S., Frank, R., Vingron, M., and Dobberstein, B. (1989) Nature 340, 478-482 [CrossRef][Medline] [Order article via Infotrieve]
  32. Shinkai, A., Mi, L. H., Tokuda, H., and Mizushima, S. (1991) J. Biol Chem. 266, 5827-5833 [Abstract/Free Full Text]
  33. Simon, S. M., and Blobel, G. (1992) Cell 69, 677-684 [Medline] [Order article via Infotrieve]
  34. Szoka, F. J., and Papahadjopoulos, D. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4194-4198 [Abstract]
  35. Teschke, C. M., Kim, J., Song, T., Park, S., Park, C., and Randall, L. L. (1991) J. Biol. Chem. 266, 11789-11796 [Abstract/Free Full Text]
  36. Ulbrandt, N. D., London, E., and Oliver, D. B. (1992) J. Biol. Chem. 267, 15184-15192 [Abstract/Free Full Text]
  37. Vaskovsky, V. E., Kostetsky, E. Y., and Vasendin, I. M. (1975) J. Chromatogr. 114, 129-141 [CrossRef][Medline] [Order article via Infotrieve]
  38. Wolin, S. L. (1994) Cell 77, 787-790 [Medline] [Order article via Infotrieve]
  39. Yi, G. S., Choi, B. S., and Kim, H. (1994) Biophys. J. 66, 1604-1611 [Abstract]

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