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J. Biol. Chem., Vol. 283, Issue 23, 15709-15715, June 6, 2008
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From the Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, December 19, 2007 , and in revised form, March 21, 2008.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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S, a poorly hydrolyzable ATP analog, or ADP plus AlF4, which mimics the transition state during ATP hydrolysis. In contrast, when SecA was in its ADP-bound state, the translocating polypeptide moved back into the cytosol, as indicated by the disappearance of the protected fragment. Backsliding was not significantly altered by deletion of the plug domain, a short helix in the center of the SecY channel, but it was slowed down when changes were introduced into the pore ring, the constriction of the hourglass-shaped channel. In all cases, backsliding was significantly slower than forward translocation. Together, these data suggest that SecA binds the polypeptide chain in its ATP state and releases it in the ADP state. The channel itself does not bind the polypeptide chain but provides "friction" that minimizes backsliding when ADP-bound SecA resets to "grab" the next segment of the substrate. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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SecA is a multidomain protein that contains two nucleotide binding folds (NBF1 and 2) with the nucleotide binding site in between them (10). ATP binding and hydrolysis are linked to conformational changes of the other domains that bind and release the polypeptide chain (5, 11–13). The SecY channel consists of the channel-forming SecY subunit and two small subunits, SecG and SecE (for review, see Ref. 1). The crystal structure of an archaeal channel homolog indicates that the pore has an hourglass shape, with a ring of 6 hydrophobic amino acids at its constriction, referred to as the pore ring (14). The pore is blocked by a central short helix, the plug, which abuts the pore ring. Channel opening moves the plug away (15, 16) and allows the passage of a translocating polypeptide through the pore ring (17). The crystal structure of the closed archaeal channel indicates that even an unfolded polypeptide could not pass through the pore ring. Thus, one has to postulate that the pore widens during translocation. The plug and the pore ring are both in close proximity to translocating polypeptides (8, 17) and could therefore provide either "friction," reducing the sliding of a polypeptide in the channel, or even bind the polypeptide as SecA resets.
Here we have addressed the mechanism of SecA-mediated translocation by studying the sliding of a polypeptide chain in the channel. To this end, we have generated translocation intermediates in which complete translocation of a polypeptide chain is prevented by a C-terminal disulfide-bridged loop that is too large to move through the channel (18). We show that these polypeptide chains move slowly back into the cytosol when SecA is in its ADP-bound state but not when it is in the ATP state or in the transition state during ATP hydrolysis. Backsliding is not greatly affected by the deletion of the plug domain from SecY, but it is significantly slowed when changes are introduced into the pore ring. These data suggest that the polypeptide is bound and pushed by SecA in its ATP state. The channel does not bind the polypeptide but provides sufficient friction to minimize backsliding when SecA resets in its ADP state.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Preparation of Proteoliposomes and Inverted Membrane Vesicles—Purified SecY mutants were reconstituted with Escherichia coli polar lipids into phospholipid vesicles as described previously (19). Inverted membrane vesicles (IMVs) were prepared as described previously (21).
Generation of proOmpA Mutants—A truncated version of proOmpA (pOA) was used (amino acids 176–297 deleted) with a cysteine introduced at the new position 202. PCR-based mutagenesis was used to introduce a cysteine at position 164 or 175 to allow for disulfide loop formation. DNA templates coding for pOA were used to make mRNA by in vitro transcription with SP6 polymerase. pOA was synthesized in the presence of [35S]methionine by in vitro translation (rabbit reticulocyte lysate; Promega) for 20 min at 30 °C, precipitated with ammonium sulfate for 30 min at 4 °C, pelleted at 14,000 rpm for 10 min at 4 °C, and resuspended in urea buffer (8 M urea, 50 mM Tris, pH 7.5).
Intermediate Formation, Backsliding, and Chase Reactions—To generate an arrested translocation intermediate, proteoliposomes containing SecY complex were mixed with in vitro translated 35S-labeled pOA and 20 µg of cysteine-free SecA (22) in buffer (50 mM KCl, 50 mM HEPES, pH 7.5, 5 mM MgCl2, 0.5 mg/ml bovine serum albumin, 0.1 mM sodium tetrathionate, and 0.2 mM ATP). After 15 min of incubation at 37 °C, a sample was taken for t = 0 and immediately digested with 1 mg/ml proteinase K. The remaining aliquot was depleted of ATP with 0.5 units of hexokinase and 10 mM glucose, and samples were removed at the times indicated and digested immediately with proteinase K. For chase incubations, 10 mM DTT was added instead of hexokinase/glucose. When indicated, 2.5 mM ADP, 1 mM ATPS, or ADP plus AlF4 (2.5 mM ADP, 5 mM NaF, and 0.3 mM AlCl3) were added to the reactions (either from the start or after intermediate formation). Samples were then incubated in 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride for 10 min at 4 °C, precipitated with 10% trichloroacetic acid for 20 min at 4 °C, pelleted at 14,000 rpm for 15 min at 4 °C, and resuspended in sample buffer (50 mM Tris, pH 7.5, 8 M urea, 5% SDS, 10 mM EDTA, 0.25 mg/ml bromphenol blue). Samples were separated on 4–20% Tris-HCl gels (Bio-Rad), visualized by autoradiography, and quantified by phosphorimaging (Fujix BAS 2000). Background subtraction and cropping of three experiments was performed with ImageGauge v4.22.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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18k-Da fragment was seen that corresponds to the 150 N-terminal amino acids of the substrate (Fig. 1A, lane 2); this corresponds to the segment of the translocation intermediate that is located inside the vesicles when the disulfide loop reaches the channel. In the presence of ATP, SecA would be expected to push the polypeptide chain continuously into the channel, thus maintaining the position of the polypeptide chain and giving rise to a defined proteolytic fragment. The intermediate did not form in the absence of ATP (Fig. 1A, lane 8) and was digested by proteinase K when detergent was added to disrupt the membrane after translocation (lane 9). When DTT was added at the beginning of the incubation to reduce the disulfide-bonded loop, only the full-length polypeptide was seen after proteolysis (Fig. 1A, lane 1; the doublet is caused by cleavage of the signal sequence by signal peptidase in IMVs). Similarly, when DTT was added after the translocation intermediate had formed, the 18-kDa fragment disappeared and full-length substrate appeared instead (Fig. 1A, lane 7). These data confirm that the 18-kDa fragment corresponds to a productive translocation intermediate that can be chased into the fully translocated polypeptide when the disulfide bridge is reduced and ATP is present.
To test the stability of the translocation intermediate in the absence of ATP, i.e. under conditions where SecA cannot push the polypeptide chain into the channel, we generated the translocation intermediate and then depleted ATP by the addition of hexokinase/glucose. Samples were taken at different time points and analyzed after treatment with proteinase K. The amounts of the 18-kDa fragment gradually decreased with time (Fig. 1A, lanes 2–6; quantitation in Fig. 1C). The overall amounts of substrate before addition of protease remained constant (supplemental Fig. S1). We interpret the disappearance of the proteolytic fragment as backsliding of the disulfide-arrested polypeptide into the cytosol; when the disulfide loop moves away from the channel, the protease can cleave at different sites, generating a heterogeneous population of protected chains that can no longer be visualized as a defined band. Our data show that backsliding occurs in the absence of ATP. A similar observation has been made with reconstituted proteoliposomes containing the yeast Sec complex and the luminal ATPase BiP: a stalled prepro-
-factor intermediate was found to slide back into the cytosol in the absence of ATP (23).
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To test whether the observed backsliding rate is dependent on proteins other than the SecY channel, we performed similar experiments with proteoliposomes containing the purified E. coli SecY complex. Again, with a disulfide bond at the C terminus between Cys-175 and Cys-202, the proOmpA substrate was only partially translocated (Fig. 1B, lane 2). When DTT was added either at the beginning of the incubation (Fig. 1B, lane 1) or after the intermediate had formed (lane 7), translocation was completed. No protease-protected material was seen if ATP were depleted before translocation (Fig. 1B, lane 8) or when detergent was present during proteolysis (lane 9). When ATP was depleted after formation of the intermediate, the amount of intermediate decreased over time with kinetics similar to that seen with IMVs (Fig. 1B, lanes 2–6; quantitation in Fig. 1C). These results show that the backsliding rate is determined by the SecY channel and not by other proteins present in crude membranes. As before, backsliding was significantly faster with a proOmpA substrate containing a disulfide loop between positions 164 and 202 (Fig. 1B, lanes 11–15; quantitation in Fig. 1C).
Next we tested whether backsliding only occurs in the absence of ATP. To this end, we first generated a translocation intermediate with reconstituted proteoliposomes, using the proOmpA substrate with cysteines at positions 164 and 202. The intermediate was isolated by sedimenting the membranes. The pellet was washed to remove residual nucleotides and resuspended. As expected, a prominent protease-resistant fragment was observed (Fig. 2A, lane 2). In the presence of ATP and DTT, this band disappeared and a fully translocated species appeared instead (Fig. 2A, lane 21). When the disulfide-bonded intermediate was incubated at 37 °C in the absence of added nucleotides, the protease-protected band disappeared as before (Fig. 2A, lanes 3–5; quantitation in Fig. 2B). Backsliding was seen in the presence of ADP (Fig. 2A, lanes 6–8) or if hexokinase/glucose were added (lanes 9–11; the identity of the smaller fragment is unclear). In contrast, in the presence of ATP, when SecA is continuously pushing the polypeptide chain into the channel, the intermediate remained stable (Fig. 2A, lanes 12–14; quantitation in Fig. 2B). When the poorly hydrolyzable ATP analog ATPS was added, a slightly larger protected fragment was observed (Fig. 2A, lanes 15–17). In the presence of ADP and AlF4, which are thought to generate a transition state of ATP hydrolysis, an additional fragment was seen that was 0.5 kDa larger than seen with ATPS (Fig. 2A, lanes 18–20; the increased intensity of the band is due to the protection of an additional labeled methionine). In both conditions, the fragments were stable over time, indicating that no backsliding occurred. It should be noted that the addition of hexokinase/glucose, ATPS, or ADP plus AlF4 at the beginning of the reaction prevented translocation, demonstrating that they effectively block SecA function (Fig. 2A, lanes 22–24). Taken together, these data show that for backsliding to occur, the ATP bound to SecA needs to be hydrolyzed.
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Proteoliposomes containing the mutant SecY complexes were active in overall translocation (Fig. 3A, lanes 9 and 17 versus wild type shown in lane 1; see also Ref. 20). When the intermediates were incubated in the absence of ATP, backsliding was slightly slower than with wild type SecY (Fig. 3A, lanes 10–13 and 18–21 versus lanes 2–5; quantitation in Fig. 3B). It thus appears that the plug has a relatively small effect on the rate at which a polypeptide chain slides inside the channel.
Another SecY feature that contacts a translocating polypeptide and could therefore determine the rate of its movement is the pore ring (14). To test the role of the pore ring residues, we mutated 4 of the isoleucines to tryptophans (4W). The introduction of these bulky and hydrophobic residues into the pore ring significantly slowed the backsliding of a translocation intermediate (Fig. 3C, lanes 10–13 versus lanes 2–5; quantitation in Fig. 3D); with the 4W mutant, the intermediate was stable for at least 1 h at 37 °C. The forward translocation rate of this mutant was somewhat lower than with wild type SecY complex (data not shown), but after 15 min the amount of translocated material was about the same (Fig. 3C, lane 9 versus lane 1). Apparently, SecA can still efficiently push the polypeptide through these channels.
We also mutated 4 pore ring residues into serines. The overall translocation of this 4S mutant was about the same as that of wild type SecY, but the backsliding rate of an intermediate was reduced (Fig. 3E, lanes 10–13 versus lanes 2–5; quantification in Fig. 3F), although not quite as dramatically as with the 4W mutant. Thus, making the pore ring residues smaller and more hydrophilic also increases the friction of a translocating chain in the channel.
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1 min, corresponding to 50–60 amino acid residues/min. The same rate was measured with even higher DTT concentrations (data not shown), indicating that disulfide bridge reduction is not rate-limiting. With a substrate containing the cysteines at positions 164 and 202, for which a faster backsliding rate had been determined (half-time 5 min; Fig. 1C), the forward translocation half-time was 1–2 min (data not shown). Similar experiments, performed with IMVs and a substrate containing cysteines at positions 175 and 202, gave a somewhat higher rate of 150 amino acids/min (supplemental Fig. S2). The higher rate was not caused by the proton motive force that can be generated with IMVs, but not with proteoliposomes, because the addition of the uncoupler carbonyl cyanide 3-chloro phenylhydrazone had no effect (supplemental Fig. S2). While our translocation rates are somewhat lower than reported in the literature (270 amino acids/min) (24), the main conclusion is that SecA-mediated forward translocation in the presence of ATP is always faster than backsliding in the absence of ATP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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S or ADP plus AlF4 compared with ATP, indicating that the polypeptide chain can move back a small distance after hydrolysis of an ATP molecule. The polypeptide chain appears to be pushed to the maximum extent into the channel in the presence of ADP plus AlF4, which mimics the transition state of ATP hydrolysis.
Our results show that while SecA is in the ADP-bound state a polypeptide can slide backward in the SecY channel. This shows that SecY does not actually bind the polypeptide chain. Nevertheless, backsliding is relatively slow. In 5–20 min, depending on the substrate, only half of the polypeptide population moves back from the channel sufficiently to be cleaved by added protease into a heterogeneous population. The backsliding rate is significantly slower than the forward translocation rates seen in the presence of ATP (50–60 amino acids in 1–2 min with purified SecY in proteoliposomes). The actual difference in the kinetics may be significantly larger because in the backsliding assay even the movement of a few amino acids will result in the loss of a defined band. Taken together, these results suggest that SecY simply provides enough friction so that the polypeptide chain does not slide back to a significant extent while SecA resets in its ADP-bound state to grab the next polypeptide segment. Both a rapid conformational change by SecA and a significant, but not too strong, interaction of SecY with the substrate are required for this mechanism to work.
The backsliding rates determined by us for the fastest substrate are about the same as those seen for prepro--factor in the yeast Sec complex (23). Although the driving force for translocation is provided in entirely different ways in bacteria and yeast (SecA pushes polypeptides through the channel, whereas the endoplasmic reticulum luminal ATPase BiP binds to the polypeptide as it emerges in the endoplasmic reticulum lumen) (23), the interaction of the channel with a translocating polypeptide appears to be conserved during evolution.
Disulfide bridge cross-linking had shown that a translocating polypeptide chain contacts residues in both the pore ring and the plug (8, 17). Our present data now suggest that the pore ring in SecY is one source of friction, because the replacement of 4 of the 6 normally occurring pore residues decreased the backsliding rate of a polypeptide in the channel. As might have been expected, introducing large, hydrophobic tryptophan residues at the narrowest point of the channel increased the friction encountered by a translocating polypeptide. Surprisingly, however, we also found that the introduction of small and hydrophilic serine residues caused increased friction. Perhaps this is caused by interactions of the hydroxyl groups of the serines with the carbonyl groups of the polypeptide backbone. Replacing 4 of the pore residues with glycines or aspartates resulted in mutant proteins that did not express well and seemed to be toxic to cells.5 It thus appears that the normally occurring pore residues (isoleucines, leucines, and valines) are optimal with respect to size and polarity. The side chains of these residues would not be expected to make strong contacts with the backbone or the side chains of the translocation substrate. In addition, these residues may have just the right size to not obstruct the movement of a polypeptide through the pore and yet still restrict the permeation of small molecules. It should be noted, however, that amino acid residues outside the actual pore ring may come in contact with the translocating chain once the channel has opened, as suggested by molecular dynamics simulations (25).
Interestingly, the forward translocation of the pore mutants was less affected than their backsliding, perhaps because SecA in its ATP form changes the conformation of SecY or exerts enough force to "push" the polypeptide chain through the altered pore ring. The plug domain is another potential source of friction encountered by a polypeptide chain in the channel. However, we found that backsliding was unaffected in the plug deletion mutants. Although in these mutants new plugs are formed, the amino acids that would contact the polypeptide chain are different from the wild type situation (20) and would not be expected to interact equally strongly with a translocating polypeptide chain.
The friction provided by SecY also appears to depend on the amino acid sequence of the translocating polypeptide located in the channel. We observed differences in backsliding rates with different segments in the channel. Interestingly, forward translocation also occurs at different rates, dependent on the amino acid sequence in the channel; hydrophobic sequences significantly slow translocation (7). Comparing fast and slow backsliding substrates, we have not seen a large difference in hydrophobicity between the polypeptide segments predicted to be inside the channel. It is therefore unclear which features of the amino acid sequence are important for the observed difference.
The comparison between crude inverted vesicles and liposomes containing the purified SecY complex indicates that the backsliding rate of a substrate is mostly determined by the SecY channel. A previous study showed that changes of the SecD/SecF levels in inverted vesicles influence the movement of a polypeptide chain both in the forward and backward direction (26). Although it is possible that SecD/SecF interacts with the translocation substrate and thereby restricts its movement, variation of the SecD/SecF levels might also indirectly affect SecA-mediated translocation. In vivo, translocation at late stages can be driven by a proton motive force. The proton motive force might slow down backsliding or replace SecA in providing the forward driving force.
Our results indicate that a one-hand model, in which a polypeptide chain is bound and released by a single binding site in SecA, is sufficient to mediate translocation. The data exclude a two-hand model in which both polypeptide binding sites are present in a single SecA molecule; in this case, one would have expected that no backsliding occurs in ADP. Our data do not strictly rule out two-hand models in which the two alternating binding sites are present in two different SecA molecules (9). They also do not exclude that the ATPase cycle of SecA could cause conformational changes in SecY that would lead to the alternating tightening and loosening of the "grip" on the translocating polypeptide. However, the grip is never strong enough to prevent backsliding, indicating that this could only be an auxiliary mechanism. The simplest model is therefore that, upon ATP hydrolysis, SecA undergoes a conformational change fast enough to grab the next polypeptide segment before significant backsliding in the channel can occur.
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FOOTNOTES |
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* This work was supported, in whole or in part, by National Institutes of Health Grant GM052586 (to T. A. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.
1 Present address: Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115.
2 Present address: Dept. of Pathology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611.
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3 To whom correspondence should be addressed: Howard Hughes Medical Inst. and Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0676; E-mail: tom_rapoport{at}hms.harvard.edu.
4 The abbreviations used are: DTT, dithiothreitol; IMV, inverted membrane vesicle; pOA, proOmpA.
5 K. J. Erlandson and T. A. Rapoport, unpublished results.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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