(Received for publication, January 18, 1996)
From the
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.
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), ()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 -helix
formation, but its mutant peptide has a low
-helix content (Yi et al., 1994; Chi et al., 1995).
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.
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
10
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.
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 -helix (
)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,
ATP
S (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.
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.
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 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 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.
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.
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.
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.
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.
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.
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.''