©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The C Terminus of SecA Is Involved in Both Lipid Binding and SecB Binding (*)

(Received for publication, August 15, 1994; and in revised form, December 20, 1994)

Eefjan Breukink (1) (2) Nico Nouwen (1) (2) (3) Anne van Raalte (1) (2) Shoji Mizushima (4) Jan Tommassen (1) (3) Ben de Kruijff (1) (2)(§)

From the  (1)Institute of Biomembranes, the (2)Department of Biochemistry of Membranes, and the (3)Department of Molecular Cell Biology, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands and the (4)School of Life Science, Tokyo College of Pharmacy and Life Science 1432-1 Horinouchi, Hachioji, Tokyo 192-03 Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Using C-terminal deletion mutations in secA, we localized the previously proposed (Breukink, E., Keller, R. C. A., and de Kruijff, B.(1993), FEBS Lett. 331, 19-24) second lipid binding site on SecA. Since removal of these residues completely abolished the property of SecA to cause aggregation of negatively charged phosphatidylglycerol vesicles, we conclude that the C-terminal 70 amino acid residues of SecA are involved in lipid-binding. The C-terminal 70 amino acid residues of SecA are important for efficient in vitro translocation of the SecB-dependent precursor of PhoE across inverted inner membrane vesicles. Moreover, in vivo studies showed that this region is essential for growth. SecB and a SecB-precursor complex were shown to inhibit the SecA-mediated lipid vesicle aggregation, suggesting that the overall acidic SecB protein binds at or near the second lipid binding site on SecA. This together with the observation that the SecA mutant protein lacking the C-terminal 70 residues had a strongly reduced ability to mediate binding of SecB-precursor complexes to inverted inner membrane vesicles demonstrates that the C terminus of SecA is also involved in SecB binding.


INTRODUCTION

Protein secretion in Escherichia coli is becoming increasingly better understood. Both genetic and biochemical approaches have led to the identification of a well defined secretion apparatus (for reviews see (1) and (2) ) in which SecA, as a dimer(3, 4) , plays a central role by coupling ATP hydrolysis to translocation and by interacting with several of the known components of this apparatus. It binds the precursor through interaction with both the signal sequence and the mature part of the preprotein(5, 6) , and it recognizes the molecular chaperone SecB or precursor-SecB complexes(7) . The integral membrane protein SecY also interacts with SecA(8) . This protein forms, together with SecE and the acidic phospholipids, the high affinity binding site for SecA at the inner membrane(9) .

The notion that acidic phospholipids play a direct role in the translocation reaction came from studies using strains deficient in the gene pgsA, which encodes an enzyme responsible for the synthesis of a precursor for the acidic phospholipids in E. coli(10) . Using these strains, it was shown that both in vivo and in vitro translocations were severely hampered (11) . Reintroduction of acidic phospholipids into inner membrane vesicles derived from these strains restored the translocation activity (12) . SecA-acidic phospholipid interactions could partly explain the role of this lipid class in translocation(13, 14) , while signal sequence-acidic phospholipid interactions are also suggested to play a direct role in translocation(15) .

Some insight into the role that SecA-acidic phospholipid interactions play in translocation was provided by the observation that the interaction of SecA with acidic phospholipids results in an increase of its ATPase activity (5) and in a conformational change within the protein(16) . It was furthermore shown that SecA could efficiently insert into model membranes containing acidic phospholipids(17, 18) . This insertion was affected by nucleotides, an observation that led to the hypothesis of a SecA binding, insertion, and deinsertion cycle (17) .

We have previously shown that SecA is able to cause aggregation of negatively charged phosphatidylglycerol vesicles(19) . This aggregation property was attributed to the existence of two different lipid binding sites present in the SecA monomer; one binding site being responsible for a more hydrophobic interaction leading to insertion of SecA into the lipid phase of the membrane, and the other binding site being responsible for a more electrostatic interaction with anionic lipids leading to a more superficial interaction of SecA with the opposing membrane.

In this study, we attempted to localize the lipid binding sites within SecA using deletion mutants. We present evidence that the second lipid binding site lies, as proposed previously(19) , within the extreme 70 C-terminal amino acid residues. We furthermore present evidence that the SecB binding site on SecA is also located within the C-terminal 70 amino acid residues and coincides with the second lipid binding site.


EXPERIMENTAL PROCEDURES

Materials

Derivatives of strain RR1(20) , harboring plasmids that encode the SecA mutant proteins C95, C72, and C53, were a generous gift from Drs. Mizushima and Matsuyama. These plasmids code for SecA proteins lacking the amino-terminal 64, 267, and 438 amino acid residues respectively(21) . These proteins will now be denominated DeltaN64, DeltaN267, and DeltaN438, respectively (Fig. 1). ATP, AMP-PNP, (^1)and proteinase K were purchased from Sigma. Protein molecular weight markers were purchased from Pharmacia Biotech Inc. Aminoethylbenzenesulfonyl fluoride (a stable water-soluble substitute for the protease inhibitor phenylmethylsulfonyl fluoride) was purchased from Calbiochem. 1,2-Dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) was purchased from Avanti. Wild-type SecA protein was purified as described (17) and stored until use at -80 °C in 50 mM Tris-HCl, pH 7.6, 280 mM NaCl, 1 mM dithiothreitol, 10% (w/v) glycerol at a concentration ranging from 2 to 3 mg/ml. PrePhoE (22) and SecB (23) were purified as described. All other chemicals were of analytical grade or better.


Figure 1: The N- and C-terminally truncated SecA proteins used in this study. The boxes indicate the scaled length of the SecA fragments. The letters next to the boxes indicate the extra amino acid residues, present at both N- and C-terminal ends as a result from the cloning procedures.



Construction of C-terminal Deletion Mutations

Standard cloning procedures were performed as described(24) . Restriction endonuclease, Klenow enzyme, T4 DNA polymerase and T4 DNA ligase treatments were carried out as suggested by the manufacturers of these enzymes (Pharmacia, New England Biolabs). Plasmid pT7-secA (25) was in separate incubations digested with SnaBI, BstXI, XcmI, KpnI, NruI, EcoRV, BglII, AatII, and MluI and ligated to a synthetic linker, dTTAATTAATTAA, with stop codons in all three reading frames. The resulting plasmids encode N-terminal SecA fragments of 831, 790, 720, 557, 502, 441, 422, 322, and 237 amino acid residues long, respectively. Depending on the frame in which the linker was inserted, the SecA fragments have an extension of 2 or 3 extra amino acid residues (Fig. 1).

For the in vivo experiments, the BamHI fragments encoding the entire secA sequences(26) , including the inserted linkers, were ligated into the BamHI site of pNEB193 (New England Biolabs), placing the (mutant) secA genes under control of the lac promoter. These plasmids were then used to transform the strains MM52 (27) and MM66 (28) to test for complementation of the specific SecA defects of these strains (see text for details).

Purification of the Mutant SecA Proteins

Derivatives of strain BL21(29) , harboring the plasmids described above, were grown at 37 °C in M9 medium (30) supplemented with 0.4% glucose and 0.2% casamino acids. When the cell culture reached an OD of 0.9-1.0, isopropyl-1-thio-beta-D-galactopyranoside was added to 1 mM final concentration, and incubation was continued for 3 h. The cells were collected and washed once in 50 mM Tris-HCl, pH 7.6, 1 mM dithiothreitol, 10% (w/v) glycerol (Buffer A), supplemented with 0.1 mM aminoethylbenzenesulfonyl fluoride, and subjected to 15 sonication cycles of 20 s at 0 °C using a tip-sonicator at 100 watts. During sonication, the temperature of the solution was never allowed to rise above 20 °C. The cell lysates were then centrifuged at 5000 times g at 4 °C for 15 min. The N-terminal SecA fragment of 831 residues(N831) appeared to be soluble, while the other N-terminal fragments were overproduced as aggregates and were present in the pellet. These aggregates were subjected to a second sonification step, pelleted, and dissolved in 8 M urea, 10 mM Tris-HCl, pH 7.6, and centrifuged at 240,000 times g for 30 min. The supernatant containing the urea-dissolved N-terminal fragments was then subjected to two fast protein liquid chromatography-Mono Q 10/10 (Pharmacia) steps, yielding proteins of >90% purity. The purified proteins were stored at -20 °C in 8 M urea, 10 mM Tris-HCl, pH 7.6, 280 mM NaCl at a concentration ranging from 1.2 to 5.3 mg/ml.

The mutant protein N790 needed an extra gel-filtration step using an fast protein liquid chromatography-Superose 6 preparation grade column (16/50, Pharmacia), equilibrated in 6 M guanidine-HCl, 10 mM Tris-HCl, pH 7.6, with a flow rate of 0.3 ml/min. This fragment was stored at -20 °C in the elution buffer at a concentration of 0.7 mg/ml.

N831 was purified from the supernatant after the two sonication steps, using the Pharmacia fast protein liquid chromatography-Mono Q 10/10 column equilibrated in Buffer A. The N831-containing fractions were pooled and ammonium sulfate-precipitated (0.40 g/ml), followed by a gel-filtration step using a Sephacryl S300 HR 16/100 column (Pharmacia), equilibrated in Buffer A, with a flow rate of 0.1 ml/min. The pooled N831 fractions were subjected to a final Mono Q 10/10 step yielding a >95% pure protein. The purified protein was stored at a concentration of 2.4 mg/ml at -80 °C in Buffer A supplemented with 280 mM NaCl. All of the fast protein liquid chromatography steps described above were performed at room temperature. The mutant proteins with N-terminal deletions were isolated as described (21) and purified according to the protocol of the urea-dissolved C-terminal deletion proteins.

ATPase Assay

The ATPase activity of the SecA mutant proteins was assayed using the method of Schiebel et al.(31) . The urea-dissolved SecA mutant proteins (5 µg) were renatured from urea at 0 °C by dilution in 50 mM Hepes-KOH, pH 8.0, 50 mM KCl, 5 mM MgCl(2), 0.5 mg/ml bovine serum albumin in the absence or presence of 4 mM ATP (total volume, 50 µl) followed by incubation for 1 h at 0 °C. For control purposes, the wild-type SecA and N831, either as native proteins or denatured by the addition of urea to a concentration of 8 M, were incubated similarly. The ATPase activity was then assayed by incubating the samples for 15 min at 37 °C. Translocation ATPase of wild-type SecA and of N831 was assayed essentially as described(5) , using urea-washed inner membrane vesicles derived from strain K003 (unc)(32) , 1.1 µg of wild-type SecA or N831, 0.8 µg of SecB, 1.6 µg of prePhoE, 4 mM ATP. The ATPase reactions were assayed in 40 mM Tris acetate, pH 8.0, 10.8 mM Mg-acetate, 28 mM K-acetate, 2 mM dithiothreitol for 15 min at 37 °C in a total volume of 50 µl.

Translocation Assay

In vitro translocation was assayed according to Kusters et al.(22) with minor modifications. Reaction mixtures (150 µl) contained 50 mM triethanolamine-OAc, pH 8.0, 50 mM KCl, 5 mM MgCl(2), 0.5 mg/ml bovine serum albumin, 4 mM ATP, 0.03 mg/ml purified SecB, and inverted inner membrane vesicles (0.43 mg of protein/ml) of E. coli strain MM52, which were incubated at 42 °C for 1 h in the presence of 12.5 mM sodium azide to abolish residual endogenous SecA activity. The residual azide concentration in the translocation mixture was below 0.25 mM. Purified [S]prePhoE (4.5 µg) dissolved in 8 M urea, 10 mM Tris-HCl, pH 8.0, was added to the reaction mixture at 0 °C, and the translocation reaction was started by the addition of varying amounts of (mutant) SecA protein, and placing the reaction mixtures at 37 °C. If required, the (mutant) SecA proteins were first renatured and incubated at 0 °C for 1 h in 50 mM triethanolamine-OAc, pH 8.0, 50 mM KCl, 5 mM MgCl(2), 0.5 mM bovine serum albumin and 4 mM ATP. At different time points, the translocation reactions were stopped by adding an aliquot (25 µl) of the reaction mixture to 2.5 µl of a 5 mg/ml proteinase K solution, previously cooled on ice. The samples were incubated on ice for 1 h and, after the addition of 0.5 µl of a 100 mM phenylmethylsulfonyl fluoride solution, they were analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography. For a 20% translocation standard, 5 µl of the reaction mixture was run on an SDS-polyacrylamide gel electrophoresis gel. The percentage of translocation was quantified by liquid scintillation counting of the rehydrated excised protein bands of dried gels.

Vesicle Aggregation

The ability of SecA and mutant SecA species to induce aggregation of large unilamellar vesicles, prepared by extrusion through 400-nm diameter filters, was tested as described previously(19) .

For determining the effect of SecB on the SecA-induced vesicle aggregation, 30 µg of SecA was preincubated for 2 min at 0 °C in 100 µl of Buffer V, supplemented with 39 µg of SecB in the presence or absence of 11 µg of prePhoE or mature PhoE (both rapidly diluted from 8 M urea upon the addition of the Buffer V, already containing the SecB protein). This leads to a molar ratio of SecA (dimer) to SecB (tetramer) to PhoE (monomer) of 1:4:2. This mixture was added to 900 µl of Buffer V containing 150 µM DOPG large unilamellar vesicles.

Binding Studies

Binding of [^14C]SecB to inner membrane vesicles derived from strain S.D.12 (33) (wild-type for translocation) was studied in Buffer B consisting of 40 mM Tris acetate, pH 8.0, 10.8 mM Mg-acetate, 28 mM K-acetate, and 2 mM dithiothreitol. 0.5 µg of prePhoE was diluted from 8 M urea in a final volume of 8 µl of Buffer B containing 0.88 µg of [^14C]SecB and incubated for 10 min at 0 °C. The SecB-prePhoE complex obtained was added to 17 µl of Buffer B containing 1.5 µg of SecA protein (wild-type or N831) and inner membrane vesicles (10.7 µg of protein). After continued incubation at 0 °C for 20 min, the inner membrane vesicles were centrifuged through a 0.5 M sucrose cushion in Buffer B at 110,000 times g at 4 °C in a Beckmann TL100 centrifuge. The pellet was resuspended in 12 µl of Buffer B and processed for liquid scintillation counting.

Miscellaneous

Protein concentrations were determined according to Bradford (34) using bovine serum albumin as a standard. Monolayer experiments were performed essentially as described(17) . SecB was ^14C-labeled by the method of reductive methylation (35) , resulting in a protein that contained 2.5 [^14C]methyl groups/SecB monomer. The labeled protein was fully active in a translocation assay as compared with unlabeled SecB (not shown). Mature PhoE was isolated from the outer membrane using a method based on trichloroacetic acid precipitation, and the pure protein was stored in 8 M urea, 10 mM Tris-HCl, pH 8.0, 150 mM NaCl. (^2)


RESULTS

Purification and Characterization of Truncated SecA Mutant Proteins

To search for the lipid binding sites on the SecA molecule, we constructed a series of deletions in the secA gene, resulting in proteins that lack parts of different length at the C terminus. Fig. 2shows the end results of the purifications of the overproduced proteins.


Figure 2: The purified N- and C-terminal SecA fragments. SDS-polyacrylamide gel electrophoresis (10%) of the purified SecA fragments. Lanes1-13 represent the proteins N237, N322, N422, N441, N502, N557, N720, N790, N831, wild-type SecA, DeltaN64, DeltaN267, and DeltaN438 stained with Coomassie Brilliant Blue. The numbers next to the gel represent the position of the molecular mass markers in kDa.



ATPase Activity of the Mutant Proteins

As a first test on the in vitro functioning of the mutant SecA proteins, the basal ATPase activities of the proteins were investigated. Because most mutant proteins had to be isolated as denatured proteins, we first investigated the effect of urea treatment on the activity of wild-type SecA and N831, which were both isolated as native proteins. For this purpose, these proteins were first denatured, followed by renaturation in the presence of ATP as described under ``Experimental Procedures.'' The endogenous ATPase activity of SecA (5) was then tested for 15 min at 37 °C. The activity of the native wild-type SecA found in this assay was comparable with previously published data (5) . The ATPase activity of the urea-denatured wild-type SecA and N831, after renaturation in presence of ATP, was only slightly lower than the activity of the native protein (Fig. 3). The endogenous ATPase activity of N831 was slightly higher than the activity of wild-type SecA. When the proteins were renatured in absence of ATP, wild-type SecA lost 45% of its native endogenous ATPase activity (26% in the case of N831). Apparently, ATP has a substantial stabilizing role during the refolding process. Interestingly, the mutant proteins N790 and N720 showed a greatly increased endogenous ATPase activity (Fig. 3) as compared with wild-type SecA. The apparently increased activity of N790 was due to the hydrolysis of ATP during the renaturation for 1 h at 0 °C in the presence of ATP (data not shown). The ATPase activity of N790 when renatured in the absence of ATP was not higher than that of wild-type SecA (Fig. 3). Apparently, this protein folds in a conformation that is active in ATP hydrolysis at 0 °C. All other proteins did not show an activity at 0 °C (data not shown). These results suggest that the C terminus of SecA is not involved in the endogenous ATPase activity of SecA. The smaller deletion mutants (Fig. 3) showed no ATPase activity.


Figure 3: ATPase activity of the C-terminally truncated SecA proteins. The ATPase activity of the mutant proteins after renaturation in presence of ATP (blackbars) and after renaturation in absence of ATP (hatchedbars). The asterisk marks the virtually increased activity of the N790 protein (see text for details). N831u and wtu represent the ATPase activity of N831 and wild-type SecA after renaturing from 8 M urea.



In Vitro Translocation Activity

The truncated SecA proteins were tested for their ability to translocate prePhoE into inverted inner membrane vesicles derived from secA mutant strain MM52. The vesicles were preincubated at 42 °C with azide to inhibit residual SecA activity. This treatment almost completely abolished the translocation activity of the MM52 vesicles when assayed for translocation activity in the absence of SecA (Fig. 4A, asterisk). Except for N831 (Fig. 4), all of the mutant proteins lacked translocation activity (data not shown). With both wild-type SecA and N831, the translocation of prePhoE was completed within 5 min after the start of the reaction (Fig. 4A). The total amount of translocated prePhoE in the presence of N831 was always lower than it was in the presence of wild-type SecA. Also the initial rate of the translocation, especially at high SecA concentrations, was lower in the presence of N831 (Fig. 4B).


Figure 4: Translocation activity of wild-type SecA and N831. The translocation activity of wild-type and N831 SecA expressed in the amount of translocated purified [S]prePhoE. A, translocation activity of wild-type SecA (circles) and N831 (triangles) at protein concentrations of 10 (filledsymbols) and 25 µg/ml (opensymbols). The asterisk marks the total translocation of prePhoE in absence of SecA. B, the initial rate of translocation (i.e. the time span 1-4 min) of wild type SecA (opensquares) and N831 (filledsquares) as a function of the concentration of the two proteins.



To gain more insight into the difference between N831 and wild-type SecA, a translocation ATPase assay was performed. The translocation ATPase activity (i.e. the ATPase activity of SecA under translocation conditions, corrected for the amount of hydrolyzed ATP in the presence of only membranes) of wild-type was 515 ± 16 pmol P(i)/(µgbulletmin), while that of N831 was 389 ± 18 pmol P(i)/(µgbulletmin) (n = 3). The membrane ATPase activity (i.e. the ATPase activity in the absence of prePhoE) did not significantly differ between wild-type SecA and N831, 395 ± 13 and 406 ± 8 pmol P(i)/(µgbulletmin) (n = 3), respectively. These results show that the difference in translocation activity between wild-type SecA and N831 is probably not caused by a difference in interaction with the inner membrane (i.e. SecY/E and the acidic phospholipids), but most probably due to a difference involving the interaction with the precursor protein.

Activity of the Truncated SecA Proteins in Vivo

To test the activity of the truncated SecA proteins in vivo, the mutant secA alleles were placed under control of the lac promoter of pNEB193. In these constructs, the expression of the (mutant) secA genes was not totally repressed during growth in absence of isopropyl-1-thio-beta-D-galactopyranoside. These plasmids were introduced in MM52, a strain carrying a temperature-sensitive secA mutation, secA51(Ts)(26) . The activity of the truncated SecA proteins was checked by determining whether they could complement the temperature-sensitive phenotype of the MM52 strain. As expected, expression of wild-type SecA could complement the mutation in MM52 (data not shown). Of the C-terminally truncated SecA proteins, only N831 and N322 could complement the mutation in MM52 (not shown), suggesting that the C-terminal 70 amino acid residues of SecA are not essential for its function. However, since SecA is active as a dimer(3, 4) , the truncated SecA proteins could potentially form heterodimers with the chromosomally-encoded temperature-sensitive mutant protein, resulting in a SecA that is sufficiently active for cell viability. Therefore, it could also be argued that heterodimer formation in the case of N831 may have obscured the possible importance of the C-terminal 70 amino acid residues of SecA for its in vivo function.

To avoid this problem, we also used strain MM66(28) , which has a secA amber mutation, in complementation assays. This strain carries a temperature-sensitive amber suppressor. When shifted to a growth temperature of 42 °C, it cannot synthesize SecA, resulting in at least a 2-fold decrease of the cellular SecA concentration every generation. Whereas expression of the wild-type SecA complemented the temperature-sensitive phenotype of this strain, N831 as well as the control vector lacking the secA gene were incapable to complement the mutation (Fig. 5). Also N322 was incapable of complementing the secA amber mutation (data not shown).


Figure 5: In vivo complementation test. Shown is strain MM66 (SecA,(28) ) carrying plasmid pNEB193, this plasmid including either the gene encoding wild-type SecA, or N831, grown at 30 °C and at the nonpermissive temperature of 42 °C.



It could be argued that the temperature of 42 °C inactivated the N831 protein. However, its in vitro translocation activity was not affected by a temperature increase to 42 °C (data not shown). Apparently the C-terminal 70 amino acid residues of SecA are important for its function.

Localization of the Lipid Binding Sites of SecA

Vesicle Aggregation Test

On the basis of the amino acid sequence of SecA and the ability to cause aggregation of negatively charged lipid vesicles, we previously proposed that the second lipid binding site on SecA is located at the extreme C terminus(19) . Therefore, we tested whether N831 could cause vesicle aggregation. Strikingly, the addition of N831 to DOPG large unilamellar vesicles did not cause a change in absorbance at 400 nm (less than 1% of the amount found for wild-type SecA), while the addition of wild-type SecA caused large increases in the absorbance at 400 nm (Fig. 6), in accordance with previous data(19) . All other C-terminally truncated SecA proteins were also inactive in inducing vesicle aggregation, while all N-terminally truncated proteins remained active in vesicle aggregation (data not shown). This result directly demonstrates that the C terminus of SecA is involved in lipid binding.


Figure 6: Aggregation activity of wild-type SecA and N831. Wild-type SecA or N831 (30 µg) were added at t = 1 min to a buffer containing 150 mM DOPG vesicles, and the change in absorption at 400 nm was followed for a further 4 min.



Monolayer Experiments

Like the wild-type SecA protein (17) all mutant proteins depicted in Fig. 1, either N- or C-terminally truncated caused a similar increase of 7-8 millinewtons/m in surface pressure of a DOPG monolayer at an initial surface pressure of 25 millinewtons/m (data not shown), suggesting that SecA is able to insert into the lipid phase of the membrane with various parts of its sequence. Therefore, the lipid-binding site that is responsible for insertion of SecA cannot be determined this way.

Localization of the SecB Binding Site of SecA

The results obtained with the mutant protein N831 are in contrast with the observations of Matsuyama et al.(21) , who did not observe a difference between N831 and wild-type SecA in the in vitro translocation of preOmpF-Lpp. These authors suggested that the C terminus of SecA is not important for the translocation activity of SecA. However, the preOmpF-Lpp precursor is SecB-independent(36) , while prePhoE, used in this study, is SecB-dependent(22) . Therefore, the C terminus of SecA may also be involved in the recognition of SecB or SecB-precursor complexes. This suggestion would lead to two predictions, i.e. 1) SecB should inhibit SecA-mediated vesicle aggregation and 2) SecB binding to the C-terminally truncated SecA, bound to inverted inner membrane vesicles, is reduced.

Effect of SecB on the SecA-induced Vesicle Aggregation

SecB indeed appeared to be capable of inhibiting the SecA-mediated aggregation of DOPG vesicles (Fig. 7). In the presence of the precursor prePhoE and SecB, this inhibition of vesicle aggregation was even more pronounced, probably due to the increased affinity of SecA for SecB-precursor complexes(7) . Mature PhoE had no effect on the inhibition of SecA-induced vesicle aggregation by SecB, demonstrating the signal sequence dependence of the SecA-SecB-precursor interaction. In the absence of SecB, prePhoE could not inhibit the SecA-induced vesicle aggregation (Fig. 7). SecB, prePhoE, and mature PhoE were not able to cause vesicle aggregation under these conditions (data not shown). These results suggest that the site responsible for the vesicle aggregation property of SecA is related to the SecB-binding site on SecA or at least that these sites are situated in close proximity. Given the negatively charged nature of the SecB-protein (pI = 3.95-4.10(23) ) the first suggestion seems more likely.


Figure 7: Effect of SecB and a SecB-precursor complex on the vesicle aggregation activity of SecA. SecA (30 µg) diluted in solely 100 µl of Buffer V (column1) was assayed for its DOPG-vesicle aggregation activity, in the presence of SecB (column2), in the presence of SecB and prePhoE (column3), in the presence of SecB and mature PhoE (column4), or in the presence of prePhoE (column5).



SecB Binding to Inner Membrane Vesicles

High affinity binding of SecB to inner membrane vesicles requires the presence of SecA and preOmpA complexed to SecB(7) . We could confirm this result in that also in the case of prePhoE, high-affinity binding of SecB to the vesicles takes place only in the presence of wild-type SecA and the precursor complexed to SecB (data not shown).

Using this assay, we observed that binding of SecB to the vesicles is reduced to background levels when N831 is present in stead of wild-type SecA (Fig. 8). Since no difference in binding of N831 or wild-type SecA to the inner membrane vesicles under identical conditions could be observed (data not shown), this strongly suggests that the SecB-binding site is located near the C terminus of SecA.


Figure 8: SecB-binding activity of wild-type SecA and N831. SecB-binding to inner membrane vesicles was assayed in the absence of SecA, in the presence of additional wild-type SecA, and in the presence of N831.




DISCUSSION

The extreme C terminus of SecA contains a cluster of four positive charges between positions 890 and 898 (26) and was therefore proposed (19) to constitute the second lipid binding site. Indeed, removal of the C-terminal 70 amino acid residues of SecA resulted in the complete loss of the ability to induce vesicle aggregation. Thus, the vesicle aggregating property of SecA can be attributed to the C terminus. However, our observations that the C-terminal 70 amino acid residues of SecA are important for efficient translocation in vitro and for maximal translocation ATPase activity and that they are essential for the cell's viability suggest, furthermore, that the C terminus is important for the cellular function of SecA. This suggestion is supported by previous results obtained by Fikes and Bassford with transposon insertion studies(37) , which led these authors to propose that the C terminus of SecA is important for recognition of the precursor.

Although the second lipid binding site was discovered by the ability of SecA to cause aggregation of negatively charged lipid vesicles, it is well possible that the C terminus is actually involved in recognition of other negatively charged compounds such as DNA, RNA, or other proteins. It has been shown that SecA recognizes and binds to its own mRNA(25, 38) , but the site of SecA responsible for this binding has been localized near the N terminus(39) . The highly negatively charged SecB protein forms another candidate to interact with the C terminus of SecA. Our observation that deletion of the C terminus of SecA results in the loss of the ability to bind SecB, demonstrates that the 70 C-terminal amino acid residues of SecA are involved in SecB binding. Two possibilities should be considered. First, it could be argued that deletion of the C terminus of SecA affects other regions of the protein involved in SecB binding. In such a situation, it is most likely that this deletion will affect the translocation of precursor proteins in general. Since the deletion does not affect the translocation of SecB-independent precursor proteins(21, 40) , this possibility seems unlikely. Therefore we favor the second possibility, namely that the C terminus of SecA is directly involved in SecB binding.

SecB was reported to interact with positively charged peptides, including polylysins(41) . Fluorescence energy transfer studies showed that the two positively charged C termini of SecA are in close proximity to each other(4) . This may imply that SecB actually interacts with the two positively charged C termini of the monomers in the SecA dimer. Therefore, there is the possibility that the data of Randall (41) are relevant for SecA binding but not for precursor binding to SecB.

Very recently, it has been reported that deletion of the C-terminal 66 residues of SecA caused a reduction in the rate of processing in vivo of the SecB-dependent precursor proteins preOmpA and maltose-binding protein, while the processing in vivo of the SecB-independent precursor pre-beta-lactamase was not affected(40) . These authors also suggested that the C terminus of SecA is involved in SecB recognition. Interestingly, it was furthermore shown that the translocation ATPase activity of the mutant SecA protein lacking the 66 C-terminal residues in presence of the SecB-independent precursor protein prePhoA-Nuc was higher as compared with the translocation ATPase activity of wild-type SecA under the same conditions(40) . This led the authors to propose that the C terminus of SecA may facilitate efficient coupling between protein translocation and rounds of ATP hydrolysis. Our observations that further deletion of the C terminus beyond residue 831 led to increased ATPase activity support this proposition.

Considering the above, we furthermore conclude that the C-terminal lipid-binding site of SecA is not important for the SecB-independent translocation pathway.


FOOTNOTES

*
This work was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO). 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.: 31-30531607; Fax: 31-30522478.

(^1)
The abbreviations used are: AMP-PNP, 5`-adenylyl-beta,-imiodiphosphate; DOPG, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol.

(^2)
E. Breukink and B. de Kruijff, manuscript in preparation.


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