(Received for publication, August 15, 1994; and in revised form, December 20, 1994)
From the
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.
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.
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.
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).
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.
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.
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, N64,
N267, and
N438 stained with
Coomassie Brilliant Blue. The numbers next to the gel
represent the position of the molecular mass markers in
kDa.
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.
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/(µg
min), while that of N831 was 389 ± 18
pmol P
/(µg
min) (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
/(µg
min) (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.
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.
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.
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).
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.
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--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.