Title: The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling
Abstract: Article15 August 1997free access The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling Franck Duong Franck Duong Dartmouth Medical School, Department of Biochemistry,7200 Vail Building, Hanover, NH, 03755 USA Search for more papers by this author William Wickner Corresponding Author William Wickner Dartmouth Medical School, Department of Biochemistry,7200 Vail Building, Hanover, NH, 03755 USA Search for more papers by this author Franck Duong Franck Duong Dartmouth Medical School, Department of Biochemistry,7200 Vail Building, Hanover, NH, 03755 USA Search for more papers by this author William Wickner Corresponding Author William Wickner Dartmouth Medical School, Department of Biochemistry,7200 Vail Building, Hanover, NH, 03755 USA Search for more papers by this author Author Information Franck Duong1 and William Wickner 1 1Dartmouth Medical School, Department of Biochemistry,7200 Vail Building, Hanover, NH, 03755 USA The EMBO Journal (1997)16:4871-4879https://doi.org/10.1093/emboj/16.16.4871 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Escherichia coli preprotein translocase comprises a membrane-embedded hexameric complex of SecY, SecE, SecG, SecD, SecF and YajC (SecYEGDFyajC) and the peripheral ATPase SecA. The energy of ATP binding and hydrolysis promotes cycles of membrane insertion and deinsertion of SecA and catalyzes the movement of the preprotein across the membrane. The proton motive force (PMF), though not essential, greatly accelerates late stages of translocation. We now report that the SecDFyajC domain of translocase slows the movement of preprotein in transit against both reverse and forward translocation and exerts this control through stabilization of the inserted form of SecA. This mechanism allows the accumulation of specific translocation intermediates which can then complete translocation under the driving force of the PMF. These findings establish a functional relationship between SecA membrane insertion and preprotein translocation and show that SecDFyajC controls SecA membrane cycling to regulate the movement of the translocating preprotein. Introduction Protein export across the bacterial inner membrane is catalyzed by preprotein translocase, a multisubunit enzyme composed of an integral membrane domain, SecYEGDFyajC, and a peripheral membrane domain, SecA (for review, see Wickner and Leonard, 1996). The essential or 'core' subunits of this enzyme, identified by genetic and biochemical studies, are the SecY, SecE and SecA proteins (Schatz and Beckwith, 1990; Akimaru et al., 1991; Duong and Wickner, 1997). Translocation of preproteins in vitro has been achieved using purified SecA and SecYEG reconstituted into proteoliposomes (Brundage et al., 1990). Translocation depends upon the energy of ATP hydrolysis by SecA (Chen and Tai, 1985; Lill et al., 1989) and is strongly stimulated by the proton motive force (PMF) across the membrane (Geller et al., 1986; Shiozuka et al., 1990). In vitro studies have dissected the translocation reaction further into distinct subreactions, leading to a working model (Schiebel et al., 1991). SecA, when in contact with a preprotein, the SecYE subunits of the translocase and acidic phospholipids (Lill et al., 1990), binds ATP and supports the translocation of 20–30 residues of the preprotein across the membrane. Hydrolysis of the bound ATP then releases the preprotein from SecA, allowing the PMF to drive further translocation. Complete translocation of the preprotein can be achieved through many such cycles of ATP-driven SecA binding, limited translocation, release from the chain and PMF-driven translocation (Tani et al., 1989; Schiebel et al., 1991; Driessen, 1992). Analysis of SecA topology during ATP-driven translocation revealed that a C-terminal 30 kDa domain of SecA undergoes repeated cycles of membrane insertion and deinsertion (Economou and Wickner, 1994; Price et al., 1996). Thus, in the absence of PMF, ATP-driven translocation may be catalyzed by repeated cycles of SecA membrane insertion and deinsertion, each insertion promoting the translocation of 20–30 residues of the preprotein across the membrane (Economou and Wickner, 1994). Cross-linking experiments showed that SecA and SecY contact the preprotein during translocation and that the 30 kDa domain of SecA is largely shielded from the lipid phase of the membrane during insertion (Joly and Wickner, 1993; Eichler et al., 1997). The SecYE domain of the translocase is sufficient to provide sites for SecA binding and insertion, to activate SecA as an ATPase and to allow some translocation (Duong and Wickner, 1997). However, the translocation that takes place at SecYE is very inefficient and results in extensive ATP hydrolysis without a proportional increase in preprotein translocation (Kawasaki et al., 1993). A more efficient preprotein translocation requires the functions of SecG (Douville et al., 1994; Hanada et al., 1994) and the members of the secD operon, i.e. SecD, SecF and YajC (Gardel et al., 1990; Pogliano and Beckwith, 1994a; Duong and Wickner, 1997). The topology inversion cycle of SecG may facilitate the cycle of SecA membrane insertion and deinsertion and thus increase the efficiency of preprotein translocation (Nishiyama et al., 1996). Though it is not known how the SecDFyajC domain of translocase facilitates translocation, SecDFyajC-depleted membranes are unable to stabilize the inserted form of SecA (Economou et al., 1995) and to use or maintain a full membrane potential (Arkowitz and Wickner, 1994). Overproduction of SecDFyajC leads to enhanced SecA membrane insertion (Kim et al., 1994; Duong and Wickner, 1997). Despite these considerable advances in our understanding of the translocation reaction, important aspects of the mechanism remain unclear. The most paradoxical aspect of the translocation mechanism concerns the contribution of SecDFyajC. Though SecDFyajC-depleted or enriched membranes have an altered SecA membrane cycle, they showed normal rates of ATP-driven preprotein translocation (Economou et al., 1995; Duong and Wickner, 1997), and SecDFyajC was only found to be critical for PMF-driven translocation (Arkowitz and Wickner, 1994). Thus, the relevance of the SecA membrane cycling was questionable since there was no apparent correlation between the effects of SecDFyajC on ATP-driven preprotein translocation and SecA membrane insertion. We have now examined the effects of SecDFyajC and SecA membrane cycling on the movement of the preprotein chain in transit across the membrane. We find that SecDFyajC allows stabilization and accumulation of specific translocation intermediates which are driven forward rapidly by the PMF to complete translocation. We show that this SecDFyajC regulation of preprotein movement occurs via regulation of SecA membrane cycling and that SecA insertion (or deinsertion) correlates with a limited forward (or backward) movement of the preprotein. These findings link the effects of SecDFyajC on SecA membrane cycling and preprotein translocation and establish a direct relationship between SecA insertion and preprotein movement. Results The accumulation of translocation intermediates requires SecDFyajC The transmembrane movement of the preprotein polypeptide chain is not a continuous process but takes place in a stepwise manner (Schiebel et al., 1991; Uchida et al., 1995). In the case of proOmpA, the most abundant translocation intermediates, termed I16 and I26, have a 16 and 26 kDa translocated domain (Tani et al., 1989; Schiebel et al., 1991). Using membranes with depleted (DF−), wild-type (DF+) or enriched (DF+++) levels of SecDFyajC proteins, we previously reported that SecDFyajC does not significantly affect the rate of ATP-driven proOmpA translocation (Arkowitz and Wickner, 1994; Economou et al., 1995). However, we find that the translocation intermediate I26 is more prominent in the wild-type or SecDFyajC-enriched membranes than in the SecDFyajC-depleted ones (Figure 1A). To examine more closely the effects of SecDFyajC on I26 formation, translocation reactions were performed at low ATP concentrations (2 μM). At this ATP concentration, SecA function becomes limiting for translocation and, therefore, translocation intermediates are more abundant (Schiebel et al., 1991). As shown Figure 1B, the formation of I26 requires SecDFyajC. In the absence of SecDFyajC, I26 is not accumulated but there is a cluster of translocation intermediates of somewhat lower molecular weight than I26. Moreover, as seen most clearly at the earlier times of the reaction, SecDFyajC seems actually to reduce the rate of translocation of full-length proOmpA. These results establish a direct involvement of SecDFyajC in the ATP-driven preprotein translocation reaction. Figure 1.SecDFyajC governs the accumulation of translocation intermediates. (A) At high ATP concentration. [125I]proOmpA (1 μg/ml; 60 000 c.p.m.) pre-mixed with unlabeled proOmpA (10 μg/ml) was pre-incubated in 100 μl of TL buffer (50 mM KCl, 50 mM MgCl2, 50 mM Tris–HCl, pH 7.9) with DTT (1 mM), BSA (200 μg/ml), SecB (48 μg/ml), SecA (5 μg/ml) and urea-stripped IMVs (100 μg/ml) from E.coli BL21 (DF+), BL325 (DF−) or BL21 pCDF (DF+++) for 2 min at 37°C. Translocation was started by the addition of 2 mM ATP and arrested after the indicated times by chilling on ice. Samples were treated with proteinase K (1 mg/ml; 15 min, 0°C), TCA precipitated and analyzed by SDS–PAGE and fluorography on a '15%' gel. [125I]proOmpA (10 and 25%) added to the reaction was loaded on the gel as a standard. (B) At low ATP concentration. Translocation was performed as described above, except that an ATP-regenerating system (5 mM creatine phosphate, 10 μg/ml creatine kinase) was added and only [125I]proOmpA (1 μg/ml; 60 000 c.p.m.) was used. Translocation was started with 2 μM ATP and arrested at the indicated time by chilling on ice. Download figure Download PowerPoint SecDFyajC stabilizes translocation intermediates and slows full translocation To examine the movement of polypeptide chains without release from the translocation site, a synthetic translocation-arrested intermediate was generated by cross-linking bovine pancreas trypsin inhibitor (BPTI) to proOmpA (Bassilana and Wickner, 1993). The large covalently folded structure of BPTI blocks translocation and thereby forms an arrested transmembrane intermediate at the point of coupling (Schiebel et al., 1991). BPTI was coupled to the cysteinyl residue 302 of proOmpA using the reversible cross-linker N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). The stably folded structure of BPTI arrested the translocation of proOmpA–BPTI (and its mature form without leader sequence) at residue 302, yielding a proteinase K-resistant domain of 29 kDa termed I29 (Figure 2A). In the presence of 2 mM ATP, SecDFyajC levels did not affect the extent of I29 formation (lanes 9 and 10). Reduction of the disulfide in the cross-linker allowed release of BPTI from proOmpA and completion of translocation (lanes 11 and 12). When the translocation of proOmpA–BPTI C302 with 5 μM ATP was examined, formation of the arrested intermediate I29 was faster in the absence of SecDFyajC (lanes 1–8). In the presence of SecDFyajC, most of proOmpA–BPTI was arrested at the I26 position. Thus, by stabilizing the translocation intermediate I26, SecDFyajC slows the forward movement of the chain. Figure 2.SecDFyajC slows proOmpA translocation and stabilizes I26 and I16. (A) Translocation of proOmpA–BPTI C302 at 5 μM ATP. Translocation reactions were as described in Figure 1A but with [125I]proOmpA–BPTI C302 (1 μg/ml; 60 000 c.p.m.), 5 μM ATP and an ATP-regenerating system (5 mM creatine phosphate, 10 μg/ml creatine kinase). Translocation was arrested after the indicated times. After 16 min, 2 mM ATP (lanes 9–12) and 10 mM DTT (lanes 11 and 12) were added and the reactions incubated further for 2 min. Samples were treated with proteinase K (1 mg/ml; 15 min, 0°C) and analyzed by '15%' SDS–PAGE and fluorography. (B) Translocation intermediate I16. Translocation reactions were performed with proOmpA–BPTI C302 and 2 μM ATP for the indicated times. After 16 min, 2 mM ATP and 10 mM DTT were added (lanes 11 and 12) and incubation was continued for 2 min. Translocation reactions were analyzed by 'high' Tris SDS–PAGE and fluorography. Download figure Download PowerPoint To determine whether SecDFyajC also affected the stability of the earlier translocation intermediate I16, translocation reactions were performed at yet lower ATP concentration (2 μM; Figure 2B). Like I26, translocation intermediate I16 could only be clearly detected in the presence of SecDFyajC, and translocation to I29 was faster in the absence of SecDFyajC. The PMF completes the translocation of the intermediates stabilized by SecDFyajC Preprotein translocation is stimulated by the PMF, which can complete the forward movement of a translocation intermediate in the absence of ATP hydrolysis (Schiebel et al., 1991; Driessen, 1992). SecDFyajC is important for the full maintenance of the PMF and thus can stimulate the rate of proOmpA translocation in the presence of a redox substrate (NADH) at low ATP concentration (Figure 3A; Arkowitz and Wickner, 1994). In the presence of PMF, however, little translocation intermediate I26 was formed even in SecDFyajC-enriched membranes (Figure 3A). The I26 which had accumulated in the presence of ATP and SecDFyajC (Figure 1B) may have been chased rapidly to fully translocated proOmpA by the PMF. To test this hypothesis directly, proOmpA translocation was performed in two steps (Figure 3B). Translocation intermediates were formed with 2 μM ATP for the indicated time, ATP was removed by apyrase, thereby blocking new initiation of translocation or any ATP-driven chain movement, and a PMF was applied. In control samples without NADH (not shown), the level of I26 formed in the first incubation was dependent on SecDFyajC as seen in Figure 1B. However, in the presence of NADH, the abundant I26 intermediates which had formed at 2 μM ATP (Figure 1B) were swept forward to yield fully translocated proOmpA (Figure 3B). Moreover, though of slightly different sizes, the level of I26 which remained was independent of SecDFyajC, as also seen in Figure 3A. Thus, by increasing the formation of translocation intermediates I16 and I26, SecDFyajC increases the translocation of proOmpA in the presence of PMF. In addition to the inability of SecDFyajC-depleted membranes to provide translocation intermediates, their inability to maintain a full PMF (Arkowitz and Wickner, 1994) may also contribute to the lower efficiency of proOmpA translocation. Figure 3.The PMF chases the I26 accumulated in the presence of SecDFyajC to fully translocated proOmpA. (A) Translocation driven by both ATP and PMF. Translocation was performed as described in Figure 1B but using non-stripped IMVs from E.coli BL21 (DF+), BL325 (DF−) or BL21 pCDF (DF+++). An electrochemical gradient was generated across the membrane by addition of 5 mM NADH, and the translocation was initiated with 2 μM ATP. Reactions were arrested at the indicated times and analyzed by '15%' SDS–PAGE and fluorography. [125I]ProOmpA (25%) added to the reaction is shown as a standard. (B) Translocation can be first driven by ATP, then by PMF. Translocation was started with 2 μM ATP and, after the indicated times, arrested by apyrase addition (20 U/ml; 2 min). NADH (5 mM) was added and the reaction incubated further for 2 min. Reactions were stopped by chilling on ice and analyzed as described in Figure 1A. Download figure Download PowerPoint Both inserted SecA and I26 are unstable without SecDFyajC In the absence of SecDFyajC, either proOmpA or proOmpA–BPTI formed a cluster of translocation intermediates ranging from 24 to 26 kDa (Figures 1B and 2A, respectively). Since SecDFyajC-depleted membranes are unable to stabilize the membrane-inserted form of SecA (Economou et al., 1995), the stability of both inserted SecA and I26 may be linked and controlled by SecDFyajC. To test this, we examined the movement of the preprotein polypeptide chain in conjunction with SecA membrane insertion, as assayed by the presence of a 30 kDa C-terminal protease-protected SecA domain (Economou and Wickner, 1994). Under the same conditions which led to the formation of I24–I26 in DF− membranes (Figure 4A, lane 1; 2 μM ATP), the SecA 30 kDa domain was largely in the deinserted state and digested by protease (Figure 4B, lane 2). In the presence of SecDFyajC, however, translocation intermediates accumulated at I26 (Figure 4A, lane 2) and SecA was in the inserted state (Figure 4B, lane 1). Since additional ATP is required for SecA deinsertion, the removal of free ATP with apyrase still allowed bound SecA with its bound ATP to insert, but 'locked' this SecA in the inserted position (Economou and Wickner, 1994). Addition of the non-hydrolyzable ATP analog AMP-PNP also promoted SecA insertion. The level of SecA 30 kDa protected fragment obtained by these treatments was independent of SecDFyajC (Figure 4B, lanes 3–6), in agreement with the finding that SecDFyajC slows the ATP-driven deinsertion rather than being essential for SecA insertion per se (Economou et al., 1995). Strikingly, after apyrase or AMP-PNP treatment, the translocation intermediates formed in the absence of SecDFyajC (I24–I26) were recovered as a unique intermediate of 26 kDa (Figure 4A, lanes 5 and 9). Thus, SecA insertion promotes the forward movement of I24 to I26, and the instability of inserted SecA in the absence of SecDFyajC is likely to be responsible for the instability of proOmpA at I26. Figure 4.(A) SecDFyajC stabilizes I26. Translocation reactions were prepared as described in Figure 1B and incubated for 15 min with 2 μM ATP (1st incubation), using non-stripped IMVs from E.coli BL325 (DF−) or BL21 (DF+). Reactions were incubated further for 2 min (2nd incubation) without any addition (lanes 1–4), with 20 U/ml apyrase (lanes 5–8) or with 2 mM AMP-PNP (lanes 9–12). After these 2 min, 5 mM NADH was added where indicated and the incubation prolonged for 3 min (3rd incubation). Reactions were stopped by chilling on ice and analyzed as described in Figure 1A. (B) SecDFyajC stabilizes the inserted form of SecA. SecA membrane insertion was monitored by the appearance of a 30 kDa protease-inaccessible domain as previously described (Economou and Wickner, 1994). Reactions in 100 μl of TL buffer contained BSA, SecB, an ATP-regenerating system and IMVs from E.coli BL21 (DF+) or BL325 (DF−) at the same concentrations as in Figure 1B. Unlabeled proOmpA (1 μg/ml) and [125I]SecA (80 000 c.p.m.; 5 μg/ml) were added. After pre-incubation (2 min, 37°C), the SecA membrane cycle was started with 2 μM ATP (lanes 1–6). After 15 min, 20 U/ml apyrase (lanes 3 and 4) or 2 mM AMP-PNP (lanes 5 and 6) was added and the reaction incubated further for 2 min. Reactions were stopped by chilling on ice, digested with trypsin (1 mg/ml, 15 min, 0°C), TCA precipitated and analyzed by '15%' SDS–PAGE and fluorography. The arrow indicates the trypsin-inaccessible 30 kDa domain of SecA. Download figure Download PowerPoint The imposition of a PMF at the end of the ATP-driven reaction stabilized I26 in the absence of SecDFyajC (Figure 4A, lane 3) and prevented the deinsertion of SecA (see Figure 5 below). In the presence of SecDFyajC, the PMF allowed a partial chase of I26 to fully translocated proOmpA (Figure 4A, lane 4) and, as previously observed (Figure 3), the level of I26 which remained after imposition of a PMF was the same in the presence or absence of SecDFyajC (Figure 4A, lanes 7 and 8). The release of the preprotein from SecA is essential to allow PMF-driven translocation, since the addition of AMP-PNP before NADH prevented the chase of I26 (Figure 4A, lane 12; Schiebel et al., 1991). Apyrase treatment, however, locked SecA in the inserted position but without such an inhibitory effect on PMF-driven translocation (Figure 4A, lane 8). Thus, the preprotein chain is probably not tightly associated with SecA after removal of the free ATP by apyrase and can translocate in response to a PMF. These results indicate that the release of the preprotein from SecA upon the hydrolysis of its bound ATP precedes SecA deinsertion, which requires additional ATP binding and hydrolysis (Economou et al., 1995). In the absence of SecDFyajC, SecA deinsertion may occur without prior release of the preprotein and thereby cause a limited reverse movement of the polypeptide chain. Figure 5.SecA deinsertion and reverse translocation in the absence of SecDFyajC. (A) Either SecDFyajC or the PMF can prevent the reverse translocation of I26. Translocation reactions were prepared as described in Figure 4A, lanes 9 and 10 (2 μM ATP for 15 min followed by the addition of 2 mM AMP-PNP). To remove nucleotides, reactions (100 μl) were layered over an equal volume of sucrose solution (0.2 M sucrose in TL buffer) and membranes sedimented by ultracentrifugation (10 min, 4°C, 73 000 r.p.m., Beckman TLA100 rotor). Sediments were resuspended in 100 μl of TL buffer and re-incubated for 2 min (1st incubation) without any additions (lanes 1 and 2), with 2 mM ATP (lanes 3 and 4) or with 2 μM ATP (lanes 5–10). After these 2 min, 5 mM NADH (lanes 7 and 8) or 2 mM AMP-PNP (lanes 9 and 10) were added and the incubation prolonged for 3 min (2nd incubation). Reactions were stopped by chilling on ice and analyzed as described in Figure 1A. (B) SecDFyajC or PMF prevent ATP-driven SecA deinsertion. Translocation reactions were prepared as described in Figure 4B, lanes 5 and 6 (2 μM ATP for 15 min followed by the addition of 2 mM AMP-PNP) using [125I]SecA (80 000 c.p.m.; 5 μg/ml) and unlabeled proOmpA (1 μg/ml). Membranes were isolated by ultracentrifugation as described above and re-incubated for 2 min without any additions (lanes 1 and 2), with 2 mM ATP (lanes 3 and 4) or with 2 μM ATP (lanes 5–8). After 2 min incubation with 2 μM ATP, 5 mM NADH (lanes 7 and 8) was added and the incubation prolonged for 3 min. Reactions were stopped by chilling on ice, treated as described in Figure 4B and analyzed by '15%' SDS–PAGE and fluorography. Download figure Download PowerPoint SecA deinsertion and backward translocation If the stability of I26 depends only on the stability of inserted SecA, it may be possible to reverse the movement of the chain by promoting SecA deinsertion. Thus, after ATP-driven translocation, SecA insertion and I26 were stabilized by addition of AMP-PNP (as in Figure 4A, lanes 9 and 10) and the nucleotides were then removed by centrifugation. The stability of I26 and inserted SecA was not affected by the centrifugation treatment (Figure 5A and B, lanes 1 and 2). Re-incubation with 2 μM ATP only led to I26 backward movement and SecA deinsertion in the absence of SecDFyajC (Figure 5A, lane 5, and B, lane 6). This destabilization was reversible since readdition of AMP-PNP after incubation with 2 μM ATP induced SecA insertion (not shown) and re-stabilization of I26 in the absence of SecDFyajC (Figure 5A, lane 9). Incubation with 2 mM ATP allowed I26 to translocate fully (Figure 5A, lanes 3 and 4). Even at this ATP concentration, some limited backward movement of I26 and SecA deinsertion were observed in the absence of SecDFyajC (Figure 5A, lane 3, and B, lane 4). Thus, in response to the ATP-driven deinsertion of SecA that occurs in the absence of SecDFyajC, the translocation intermediate I26 undergoes a backward movement. The presence of SecDFyajC prevents SecA deinsertion and stabilizes I26 against reverse translocation. Imposition of a PMF during the re-incubation with 2 μM ATP chased I26 to fully translocated proOmpA in the presence of SecDFyajC (Figure 5A, lane 8) and prevented the backward movement of I26 which was seen previously in the absence of SecDFyajC (lane 7). Under these same conditions, the PMF also prevented the ATP-dependent SecA deinsertion that occurred in the absence of SecDFyajC (Figure 5B, lanes 6 and 8). Thus, like SecDFyajC, the PMF stabilizes the inserted form of SecA and prevents the backward movement of the chain. This result suggests that the stability of the chain is not directly dependent on SecDFyajC but rather on the inserted form of SecA. SecDFyajC stabilizes arrested translocation intermediates at position I26 To test whether the function of SecDFyajC is restricted to low ATP concentrations (2 μM) or whether it can also stabilize preproteins which are blocked in their forward translocation at I26 at physiological levels of ATP (2 mM), the cysteine residue C302 was relocated genetically to residue 245 and coupled to BPTI. The translocation intermediate formed with proOmpA–BPTI C245 (and its mature form without leader sequence) arrested near position I26 in the presence of SecDFyajC but at earlier translocation intermediates (ranging from I24 to I26) in its absence (Figure 6A, lanes 1 and 2). Under these same conditions, SecA was inserted in the presence of SecDFyajC and deinserted in its absence (Figure 6B, lanes 1 and 2). Addition of AMP-PNP to the reaction promoted SecA insertion as well as forward movement of the chain in the absence of SecDFyajC (Figure 6A and B, lanes 4). To determine if the inserted SecA was responsible for the stability of proOmpA–BPTI C245 at position I26, membranes were treated with urea. Such treatment extracted most of the inserted form of SecA from the membrane (Figure 6B, lanes 5 and 6), and the translocation intermediate was recovered at positions I24–I26 in either the presence or absence of SecDFyajC (Figure 6A, lanes 5 and 6), much as seen without urea extraction in the absence SecDFyajC (Figure 6A, lane 2). Thus, in the absence of inserted SecA, the stable positions of translocation intermediates are not governed by SecDFyajC. These experiments, using high ATP concentrations and intermediates where the chain is arrested near position I26, confirm that it is the SecDFyajC-mediated stabilization of inserted SecA which prevents the backward movement of the chain. Figure 6.SecDFyajC prevents the backward movement of proOmpA–BPTI C245 via stabilization of inserted SecA. (A) Translocation reactions (100 μl), as described in Figure 1A, used [125I]proOmpA–BPTI C245 (lanes 1–6; 1 μg/ml; 60 000 c.p.m.) or [125I]proOmpA-BPTI C302 (lanes 7–10; 1 μg/ml; 60 000 c.p.m.) and 2 mM ATP. After 15 min at 37°C, reactions were chilled on ice. An equal volume of TL buffer (lanes 1–4, 7 and 8) or 10 M urea (lanes 5, 6, 9 and 10) was added. After 5 min on ice, membranes were sedimented through a sucrose solution as described in Figure 4A and resuspended in 100 μl of TL buffer on ice. Reactions corresponding to lanes 3 and 4 were incubated further for 2 min at 37°C with 2 mM AMP-PNP. Samples were treated with proteinase K (1 mg/ml; 15 min, 0°C) and analyzed by '15%' SDS–PAGE and fluorography. (B) The position of the arrest of proOmpA–BPTI C245 depends on inserted SecA. Translocation reactions were as above but using [125I]SecA (80 000 c.p.m.; 5 μg/ml) and unlabeled proOmpA–BPTI C245 (lanes 1–6; 1 μg/ml) or proOmpA–BPTI C302 (lanes 7–10; 1 μg/ml). Reactions were digested with trypsin (1 mg/ml; 15 min, 0°C) and analyzed by '15%' SDS–PAGE and fluorography. Download figure Download PowerPoint While the stability of I26 depends on SecDFyajC, the stability of the translocation intermediate I29 formed using proOmpA–BPTI C302 is independent of SecDFyajC (Figures 2A, and 6A, lanes 7 and 8). However, SecA insertion was still stabilized by SecDFyajC (Figure 6B, lanes 7 and 8). Moreover, after urea treatment, most of the SecA was extracted from the inserted position but the chain remained at position I29 (Figure 6A and B, lanes 9 and 10). Thus, the stability of the chain at positions other than I26, such as I29 or I24, is not due to the inserted form of SecA and, consequently, is independent of SecDFyajC. Discussion SecD, SecF and YajC, the proteins of the secD operon (Gardel et al., 1990), are co-expressed (Pogliano and Beckwith, 1994b) and form a complex which can be isolated along with SecYEG as a 'holoenzyme' form of the translocase (Duong and Wickner, 1997). In vivo studies have shown that strains with mutations or depletion of SecDF have export defects, while their overexpression stimulates translocation and can suppress leader peptide mutations (Pogliano and Beckwith, 1994a). Addition of anti-SecD antibodies to spheroplasts induced accumulation of translocation intermediates and blocked their subsequent release into the periplasm (Matsuyama et al., 1993). While clearly establishing the importance of SecD and SecF for translocation, these in vivo studies did not define their mechanism of action. Furthermore, in vitro studies with inner membrane vesicles (IMVs) from strains with depleted or overproduced SecDFyajC showed very little alteration in translocation rates (Arkowitz and Wickner, 1994; Economou et al., 1995; Duong and Wickner, 1997), and ATP-driven translocation of proOmpA into SecYEG-reconstituted proteoliposomes showed a rate of translocation close to that observed with intact membranes vesicles (Bassilana and Wickner, 1993). Thus, the contribution of the members of the secD operon to the translocation process remained largely undefined. We have now resolved this apparent discrepancy between the genetic and biochemical studies by developing more refined biochemical assays which can assess the function of SecDFyajC in translocation. Through these assays, t