Secretion Monitor, SecM, Undergoes Self-Translation Arrest in the Cytosol

Molecular Cell, Vol. 7, 185–192, January, 2001, Copyright 2001 by Cell Press

Secretion Monitor, SecM, Undergoes Self-Translation Arrest in the Cytosol Hitoshi Nakatogawa and Koreaki Ito* Institute for Virus Research and CREST Japan Science and Technology Corporation Kyoto University Kyoto 606-8507 Japan

Summary The product of the Escherichia coli secM gene (secretion monitor, formerly gene X), upstream of secA, is involved in secretion-responsive control of SecA translation. In wild-type cells, SecM is rapidly degraded by the periplasmic tail–specific protease. It is also subject to a transient translation pause at a position close to the C terminus. The elongation arrest was strikingly prolonged when translocation of SecM was impaired. SRP was not required for this arrest. Instead, the nascent SecM product itself may participate, as the arrest was diminished when it incorporated a proline analog, azetidine. We propose that cytosolically localized nascent SecM undergoes self-translation arrest, thereby enhancing translation of secA through an altered secondary structure of the secM-secA messenger RNA. Introduction Protein translocation across a membrane is a fundamental cellular process, which is facilitated by proteinaceous components in the membrane as well as in the cytosol. In the bacterial translocation system, either SecB or SRP handles the targeting step (Fekkes and Driessen, 1999). The SecB pathway is for protein export to the periplasm, whereas the SRP pathway seems to be used for membrane protein integration. The SecA ATPase interacts with SecB, a preprotein, and the SecYEG membrane components (Duong et al., 1997; Economou, 1998). It is used, at least partly, for the SRPtargeted pathway as well (Valent et al., 1998). The expression of SecA is modulated according to the cell’s ability to export proteins; reduced ability of protein export results in upregulation of SecA translation (Schatz and Beckwith, 1990; McNicholas et al., 1997; Oliver et al., 1998). The gene called X (secM, hereafter) is located in the upstream of secA in the same operon (Schmidt et al., 1988). The secM gene product was thought to be exported to the periplasmic space, although it has only been identified as a fusion protein with alkaline phophatase (Rajapandi et al., 1991). Oliver et al. (1998) found that a signal sequence mutation of SecM led to an elevated translation of secA that was placed in cis configuration. Moreover, this effect was suppressible by a prlA mutation, an alteration in the SecY translocase subunit, that suppresses mutational defects of signal sequences. These results indicate that * To whom correspondence should be addressed (e-mail: kito@ virus.kyoto-u.ac.jp).

the export status of SecM affects translation of SecA encoded in the same messenger RNA strand. The cisspecific effect suggests that the SecM translocation includes some cotranslational event, which has only been ill-defined in prokaryotes. The potential usefulness of SecM in studying a cotranslational protein translocation mechanism prompted us to characterize it. As the product of secM had only been identified as a fusion protein with alkaline phosphatase (Rajapandi et al., 1991), we intended to identify the native SecM by preparing and using polyclonal peptide antibodies. Our characterization of the secM gene product revealed unusual properties of SecM. First, it is very unstable when exported to the periplasmic space, arguing against the notion that it carries out some physiological functions in this location. Second, translation of secM was found to be subject to a pause, which is enhanced strikingly when translocation of SecM was retarded. Our results suggest that nascent chain of SecM has unique properties of arresting its own translation when it is localized in the cytosol. Results SecM Is Degraded in the Periplasm by the Tail-Specific Protease In our initial attempts to identify the secM gene product, it was expressed from a plasmid and its biosynthesis was followed by pulse–chase and immunoprecipitation experiments. In wild-type cells, a protein species of about 15 kDa (band A) was initially labeled and then disappeared within 2 min (Figure 1A, lanes 1–5). As shown below, band A proved to represent a translationpause product of SecM. In a strain deleted for prc, encoding the tail-specific protease in the periplasm (Silber et al., 1992; Hara et al., 1996), a partially stabilized band of about 13 kDa appeared (Figure 1A, lanes 11–15; band M). Band M protein was recovered from the periplasmic fraction upon cell fractionation (data not shown). These results suggest that band M was the exported mature product of SecM, which was degraded by the tail-specific protease (Silber et al., 1992; Keiler et al., 1996) in the wild-type periplasm. SecM contains only one methionine in the mature sequence. We attached hexa methionine sequence (Met6) to the C terminus of SecM (SecM-Met6) to enhance its labeling with [35S]methionine. We also anticipated that addition of Met6 at the C terminus might make SecM resistant to the tail-specific protease. When SecM-Met6 was expressed in wild-type cells, a protein (M⬘ in Figure 1A, lanes 16–20) of slightly higher molecular mass than that of band M was labeled intensely. Band M⬘ was stable even in the prc⫹ strain (Figure 1A, lanes 16–20) and fractionated as a periplasmic protein (data not shown). These results show that, although SecM is designed to be exported to the periplasmic space, it is immediately eliminated by the tail-specific protease in this location. Since stabilization was not complete in the ⌬prc strain (Figure 1A, lanes 11–15), some other proteases may also contribute to this degradation.

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Figure 1. Synthesis and Stability of the secM Gene Products In Vivo (A) SecM and SecM-Met6 synthesized in wild-type, secY-defective, and prc-defective cells. SecM (on pNH21; lanes 1–15) or SecM-Met6 (on pNH22; lanes 16–25) was induced for 30 min either in “wild-type” strain (GN40/pSTD343; lanes 1–5 and 16–20), secY24/Syd strain (Shimoike et al., 1995) (KI297/pST30; lanes 6–10 and 21–25), or prc⌬::neo strain (Hara et al., 1996) (JE7934; lanes 11–15) and subjected to pulse–chase and immunoprecipitation analyses. Note that Syd also was induced in the secY24/Syd strain, resulting in severely impaired SecY function (Matsuo et al., 1998). Band A represents the translation-arrested fragment. M and M⬘ represent the mature forms of SecM and SecM-Met6, respectively. P⬘ represents the full-length precursor form of SecM-Met6. (B) The band A product lacks the C-terminal region. SecM-Met6 (on pNH22; lanes 1–5) and SecM-HA-Met6 (on pNH23; lanes 6–15) were analyzed by pulse–chase experiments. Samples were immunoprecipitated with either anti-SecM (lanes 1–10) or anti-HA (lanes 11–15). M⬘⬘ and P⬘⬘ indicate the products similar to M and P but retaining both Met6 and HA sequences.

SecM Lacking the C-Terminal Region Is Produced Transiently (secⴙ Conditions) or Stably (sec⫺ Conditions) When the SecY function was compromised by a combination of the secY24 mutation and Syd overproduction (Shimoike et al., 1995; Matsuo et al., 1998), the band A product of SecM was stabilized strikingly (Figure 1A, lanes 6–10). When SecM-Met6 was expressed under the SecY-defective conditions (Figure 1A, lanes 21–25), the band M⬘ product was not seen, consistent with its being exported mature product. Again, the band A product was stabilized markedly. In addition, a new band, P⬘, appeared. Band A and band P⬘ proteins were intracellular (data not shown). Band P⬘ product may have been the precursor form of SecM-Met6, which became detectable because of the increased labeling due to Met6. Similarly, the attachment of Met6 increased the extent of [35S]methionine labeling as well as the molecular mass of the mature products (Figure 1A, compare M and M⬘ for lanes 11–15 and lanes 16–20). Interestingly, neither intensity nor electrophoretic mobility differed significantly between band A products of SecM and SecM-Met6 (Figure 1A). This raised a possibility that band A lacked the C-terminal region. The absence of the C-terminal region in band A was shown conclusively by comparing SecM-Met6 and SecM-HA-Met6, in which the HA epitope sequence was inserted between SecM and Met6. In the latter construction, the bands M⬘⬘ and P⬘⬘, corresponding to M⬘ and

P⬘, were upshifted due to the addition of HA (Figure 1B, compare lanes 1 and 6), but band A remained unchanged in electrophoretic mobility (Figure 1B, lanes 1 and 6). Furthermore, band A did not react with anti-HA, whereas bands M⬘⬘ and P⬘⬘ did (Figure 1B, lanes 11–15). In contrast, insertion of HA into an N-terminal region of mature sequence of SecM (SS-HA-SecM-Met6) upshifted all the bands, including the one corresponding to band A (see Figure 2A, lanes 13 and 14). Thus, band A contains the N-terminal region but not the C-terminal region of SecM. These results show that SecM is produced as a species lacking the C-terminal region either transiently in wild-type cells or stably in the SecY-defective cells. The secM Translation Is Subject to Elongation Pause The SecM species lacking the C-terminal region may have been produced either by proteolytic cleavage or by incomplete translation. To discriminate between these possibilities, an SDS extract from pulse-labeled cells expressing SecM-Met6 was treated with cetyltrimethylammonium bromide (CTABr), a reagent that precipitates nucleic acids (Gilmore et al., 1991). Band A was predominantly recovered in the precipitates (Figure 2A, lane 5), whereas bands M⬘ and P⬘ were exclusively in the supernatant (lane 6). When the sample was treated with RNase before CTABr precipitation, band A was no longer recovered from the precipitates (Figure 2A, lanes

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Figure 2. Translation-Arrested and tRNAAttached Forms of SecM Are Produced Both In Vivo and In Vitro (A) CTABr fractionation of in vivo products. SecM-Met6 (on pNH22; lanes 1–8), SecM (on pNH21; lanes 9 and 10), SecM-HA-Met6 (on pNH32; lanes 11 and 12), and SS-HA-SecMMet6 (on pNH31; lanes 13 and 14) were pulse labeled with [35S]methionine for 1 min and CTABr fractionated. Samples for lanes 3, 4, 7, and 8 were incubated with 100 ␮g/ml RNase A (at 37⬚C for 10 min) before the fractionation. Samples were subjected to SDSPAGE either directly (lanes 1–4; sample sizes for lanes 2 and 4 were 1/10) or after immunoprecipitation (lanes 5–14). P and S indicate the pellet and the supernatant, respectively. A⬘⬘ indicates the translation-arrested product with the extra HA sequence after the signal sequence. (B) Translation arrest in vitro. In vitro synthesis of SecM-Met6 was directed by pNH1 DNA (0.5 ␮g), in the presence of 45 ␮l of transcription-translation mixture (Baba et al., 1990), containing S140 extract from strain AD202 (Matsumoto et al., 1997), T7 RNA polymerase (45 units), and [35S]methionine. During incubation at 37⬚C, 10 ␮l samples were withdrawn for immunoprecipitation and SDS-PAGE (lanes 2–5). The 10 min sample was also CTABr fractionated (lanes 6 and 7). Lane 1 received an in vivo sample for comparison.

7 and 8). Thus, band A product was still attached to an RNA molecule. We conclude that it represents an elongation-arrested peptidyl-tRNA molecule. CTABr precipitability was confirmed for band A or its equivalents for wild-type SecM (Figure 2A, lane 9), SecM-HA-Met6 (Figure 2A, lane 11), and SS-HA-SecM-Met6 (Figure 2A, lane 13). When SecM-Met6 was synthesized in vitro using Escherichia coli coupled transcription-translation system, a product corresponding to band A was initially produced and then converted to the full-length P⬘ product upon further incubation (Figure 2B, lanes 2–5). The in vitro– produced band A was precipitable by CTABr (lane 6). Thus, translation of secM is subject to pause even in vitro. These results establish that the secM translation is subject to a pause at a position close to its C terminus, a possibility that was pointed to before (Oliver et al., 1998) but has never been examined. SecM Translation Arrest Is Exaggerated under Translocation-Defective Conditions As already shown in Figure 1A, an impairment of the SecY function led to a striking stabilization of the translation-arrested forms of SecM (lanes 6–10) and SecMMet6 (lanes 21–25), which persisted up to 60 min examined (data not shown for extended chase experiments). Less striking but significant enhancement in translation arrest of SecM-Met6 was also observed when it was expressed in the secY39, secY40 (Baba et al., 1990), and ⌬secG (Nishiyama et al., 1994) mutant cells (data not shown). Striking prolongation of the translation arrest was observed also when cells were treated with sodium azide, an inhibitor of SecA, before pulse labeling (Figure 3A, lanes 6–10). Thus, the translation arrest becomes more efficient and long-lasting when the functions of cellular translocation machinery were compromised. It was reported that overproduction of FtsY, the E. coli homolog of the signal recognition particle (SRP)

receptor (Walter and Johnson, 1994), causes disturbance of the SRP system in E. coli (Luirink et al., 1994). Under such conditions, SecM was stabilized markedly (Figure 3B, lanes 6–10). Expression of the dominantnegative variants of FtsY (Kusters et al., 1995) also caused similar stabilization of the arrested state of SecM (data not shown). It should be noted that some of the above conditions, such as the secY40 mutation (Baba et al., 1990) and FtsY overproduction (Luirink et al., 1994), do not significantly impair translocation of typical exported proteins like OmpA, as we confirmed in this study (data not shown). We reason that the translocation state of SecM itself is crucial for the establishment of translation arrest. A signal sequence mutation in secM was reported to enhance secA translation in a cis-specific manner (Oliver et al., 1998). We examined a SecM-Met6 derivative having the same signal sequence mutation (⌬LGLPA-SecM-Met6). This mutant protein showed prolonged translation arrest even when it was expressed in wild-type cells (Figure 4A, lanes 6–10; see lane P for the CTABr precipitability). Thus, not only the defects in the translocation machinery but also the defects in the cis-translocation element result in the prolonged translation arrest of SecM. Neither Signal Sequence Nor SRP Is Required for the Translation Arrest The eukaryotic SRP has an activity to arrest translation when it binds to the signal sequence of nascent secretory protein in the absence of the SRP receptor (Walter and Johnson, 1994). To examine whether the E. coli SRP has a role in the translation arrest of SecM, we used a strain in which expression of Ffh, the signal sequence binding protein component of E. coli SRP, is placed under the araB promoter (Phillips and Silhavy, 1992). When this strain carrying a SecM-Met6 plasmid was cultured in the presence of arabinose, band A product was extremely unstable (half-life, 0.5 min; Figure 3C, lanes

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Figure 4. Defective as well as Deleted Signal Peptides Lead to Prolonged Translation Arrest

Figure 3. Defects in SecA and SRP Lead to Prolonged Translation Arrest Cells were pulse labeled with [35S]methionine for 1 min and then chased for 0 (lanes 1 and 6), 1 (lanes 2 and 7), 2 (lanes 3 and 8), 4 (lanes 4 and 9), and 8 (lanes 5 and 10) min. (A) SecM-Met6 was examined by pulse and chase in the presence (lanes 6–10) or the absence (lanes 1–5) of 0.02% sodium azide added 1 min before pulse labeling, followed by anti-SecM immunoprecipitation. (B) SecM, under SRP-SRP receptor system-disturbed conditions, was examined using strain JM109(DE3) carrying pNH26 alone (lanes 1–5) or both pNH26 and pET9-FtsY (FtsY-overproducing plasmid; lanes 6–10) (Luirink et al., 1994). (C) SecM-Met6 (on pNH5) was examined in strain WAM113 (Phillips and Silhavy, 1992), in which ffh had been placed under the control of the araB promoter. Cells were grown first in M9 medium supplemented with 0.2% arabinose, washed three times with M9 salts, and inoculated (inoculum size, 1/500) into two portions of M9-amino acids media, one containing 0.2% arabinose (lanes 1–5 and A) and another containing 0.4% glucose (lanes 6–10 and G). After shaking at 37⬚C for 7 hr (with appropriate dilutions), SecM-Met6 was induced for 15 min and pulse–chase experiments were performed (lanes 1–10). Quantification of band A indicated that its half-life was 0.5 min for the arabinose-grown cells and 2 min for the Ffh-depleted cells. Ffh content was examined by immunoblotting (lanes A and G) using peptide antibodies against residues 419 to 432 of Ffh.

1–5). After cultivation of the same cells in the absence of arabinose and in the presence of glucose, cellular Ffh content dropped 10-fold (Figure 3C, compare lanes A and G). Under the latter conditions, the half-life of band A was prolonged about 4-fold (Figure 3C, lanes 6–10). Thus, the lack of SRP function leads to enhanced SecM translation arrest. This is consistent with the notion that SRP is required for translocation of SecM but inconsistent with the notion that SRP is a component required for the arrest. To examine whether signal sequence is at all required for the arrest, we constructed ⌬SS-⬘SecM-Met6 that lacked the N-terminal 48 residues of precursor SecM including the entire signal sequence (see Sarker et al., 2000 for the updated signal sequence of SecM). This mutant form of SecM-Met6 was synthesized largely as the arrested form (Figure 4B, lane P for CTABr precipitability), which disappeared only very slowly (Figure 4B, lanes 6–10). Thus, enhanced translation arrest occurred in the total absence of signal sequence. These results

Cells were pulse labeled with [35S]methionine for 1 min and then chased for 0 (lanes 1 and 6), 1 (lanes 2 and 7), 2 (lanes 3 and 8), 4 (lanes 4 and 9), and 8 (lanes 5 and 10) min. (A) ⌬LGLPA-SecM-Met6 (on pNH30) was examined by pulse–chase and immunoprecipitation (lanes 6–12). The 0 min chase sample was CTABr fractionated (lanes P and S). Lanes 1–5 were for a control experiment using SecM-Met6. A* indicates the translation-arrested fragment of ⌬LGLPA-SecM-Met6. (B) ⌬SS-⬘SecM-Met6 (on pNH7) was examined as in (A). A** indicates the translation-arrested fragment of ⌬SS-⬘SecM-Met6.

establish that the SecM translation arrest is mechanistically distinct from the SRP-mediated translation arrest observed in eukaryotes (Walter and Johnson, 1994). We surmise that whenever the nascent SecM product is localized in the cytosol, it undergoes self-translation arrest. Nascent SecM Participates in the Elongation Arrest Translation pause and its modulation by the translocation status of the nascent chain pose intriguing questions as to the molecular mechanisms responsible for this unusual regulation. It is easily conceivable that some features of messenger RNA are involved in the elongation pause (Wolin and Walter, 1988; McNicholas et al., 1997). In addition, the nascent product may have a role in establishing the prolonged elongation arrest (see Discussion). Mutational studies on such a role of the nascent product are associated with intrinsic drawbacks, since any mutation introduces an alteration into messenger RNA as well as into protein. Thus, it is difficult to conclude whether the mutational effect is due to the protein alteration or the messenger RNA alteration. To circumvent this difficulty, we used an amino acid analog, which is incorporated into proteins, to alter the presumed functionality of the nascent chain. Either proline or its analog, azetidine, was added to the culture 3 min before pulse labeling SecM-Met6. It is unlikely that any components of translation machinery are affected in their activities by this brief exposure to the amino acid analog. The apparent protein synthesis rate, as examined by incorporation of [35S]methionine, declined somewhat (by 20%–30%) under these conditions. Under the SecY-defective conditions, the elongation-arrested form of SecM-Met6 was the major product in the presence of proline (Figure 5, compare lanes 1 and 9 for materials recovered in CTABr precipitates and superna-

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Figure 5. Azetidine Incorporation Cancels the Enhanced Translation Arrest Strain KI297/pST30 (secY24/Syd) harboring pNH22 (SecM-Met6) was grown in M9 medium supplemented with 17 amino acids (20 ␮g/ml each, other than Met, Cys, and Pro). SecM-Met6 and Syd were induced for 25 min, and then 100 ␮g/ml of either L-proline (lanes 1–4 and 9–12) or 2-azetidinecarboxylic acid (lanes 5–8 and 13–16) was added. Syd overproduction causes a translocation defect in the cells. Three minutes later, cells were subjected to pulse–chase and CTABr fractionation (lanes 1–8, precipitates; lanes 9–16, supernatant), followed by SecM immunoprecipitation.

tant, respectively). It persisted during the chase period with only slow decrease (Figure 5, lanes 1–4). In contrast, the major product labeled in the presence of azetidine was the full-length P⬘ form of SecM-Met6 seen in the CTABr supernatant (Figure 5, compare lanes 5 and 13). The arrest product in the azetidine-treated cells disappeared much more rapidly (Figure 5, lanes 5–8) than that produced in the presence of proline (lanes 1–4). Azetidine treatment did not affect protein export status of either sec⫹ or secY⫺ cells (Figure 5 and data not shown). These results suggest that the nascent product has a positive role in establishing the long-lasting elongation arrest. Presumably, this activity was lowered by azetidine substitution for some of the 13 proline residues present in the SecM precursor molecule. Sec Defect Responses of the Chromosomal secM-secA Expression The results so far presented are concerned with SecM that was expressed from multicopy plasmids in the absence of cis-located secA gene. We examined whether the translation pause mechanism is operating in the chromosomal secM-secA configuration and how it responds to the translocation defect caused by the SecY deficiency (Figure 6). Wild-type and the secY24 mutant

Figure 6. Secretion Defect-Response of the Chromosomal secMsecA Expression (A) Wild-type cells (KI298/pSTV29, Shimoike et al., 1995; lane 1) and the SecY-defective cells (KI297/pST30; lane 2) were induced with IPTG for 25 min and pulse labeled with [35S]methionine for 2 min. In the latter cells, Syd overproduction blocked the protein translocase. Samples were subjected to CTABr precipitation followed by SecM immunoprecipitation (bottom). They were also subjected to OmpA immunoprecipitation (middle) and SecA immunoprecipitation (top). Equal radioactivities were used between the two strains, but relative amounts used for CTABr precipitation, OmpA immunoprecipitation, and SecA immunoprecipitation were 60:1:2, respectively. (B) Relative radioactivities associated with SecA (filled column) and the arrest fragment of SecM (open column) are compared between the two strains. Values for the wild-type strain were set as unity.

cells with overproduced Syd were pulse labeled with [35S]methionine and divided into three portions. One was CTABr precipitated and then immunoprecipitated with anti-SecM. The other two samples were directly immunoprecipitated with anti-OmpA, to monitor protein secretion status, as well as with anti-SecA, to assess the extent of SecA derepression. We were able to detect the arrested fragment of SecM as it was expressed from the chromosomal gene (Figure 6A, bottom). Its intensity increased markedly in the SecY-compromised cells (Figure 6A, bottom, lane 2), in which OmpA processing was almost completely blocked (Figure 6A, middle, lane 2). As expected, the synthesis of SecA was elevated markedly in the latter cells (Figure 6A, top, compare lane 2 with lane 1). The degree of Sec defect–dependent increase in the arrested SecM band roughly correlated with that of SecA biosynthesis (Figure 6B).

Discussion There has been no report on identification of the secM gene product, except for the forms of fusion proteins with alkaline phosphatase (Rajapandi et al., 1991). Thus, it has only been inferred that SecM is an exported protein, as it contains presumed signal sequence and it is able to export attached alkaline phosphatase moiety to the periplasm. Our attempts to identify the product of secM by immunoblotting also failed (data not shown). Thus, this gene encodes a peculiar protein, which does not accumulate significantly in the normal E. coli cells. Our results show that this is partly because SecM is rapidly degraded in the location of its final destination, the periplasmic space. The tail-specific protease is mainly, but not exclusively, responsible for this degradation. The instability of SecM in the periplasm argues against the notion that it carries out some physiological function in this location. Probably, its sole function in the cell is to monitor protein translocation activity and thereby to modulate the expression level of SecA at the translation level (Oliver et al., 1998). The secM and secA genes form a single transcription unit (Schmidt et al., 1988). Previous studies by Oliver and coworkers showed that translation of SecA is modulated by multiple mechanisms. First, the basal secA expression is translationally coupled with the translation of the upstream secM gene (Schmidt et al., 1988; Fikes and Bassford, 1989). Second, SecA may act as an autogenous repressor of its own translation (Schmidt and Oliver, 1989; Salavati and Oliver, 1997). Finally, export status of SecM affects translation of secA in cis-specific

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manners (Oliver et al., 1998). This last property of SecM seems to provide a key mechanism for the secretionresponsive feature of secA expression. In addition, it is a strong and possibly the sole piece of genetic evidence suggesting that a cotranslational protein translocation mechanism exists in E. coli, at least for SecM. We have shown here that in cells of normal translocation activity, SecM transiently accumulates as a form of elongation-arrested peptidyl-tRNA, which disappears with a half-life of 1 min or less (Figure 1A). We do not know the exact fate of this product. Probably a major fraction may be converted to the full-length and exported product, which is then degraded rapidly. The result presented in Figure 1A, lanes 16–17, indicates that the initial proportion of the translation-pause product was at least 50% (note that the radioactive intensities should be corrected for the different numbers of methionine for band A and band M⬘). However, the data do not show a simple precursor–product relationship for band A and band M⬘. We believe that this was partly because mature SecM-Met6 was still degraded slowly. It is also conceivable that some fraction of the arrested product was degraded in the cytosol. In the absence of active translocation, the elongation arrest became strikingly prolonged. However, both the elongation-arrested fragment and the full-length precursor protein seen under the SecY-defective conditions (Figure 1A) or in the presence of the signal sequence mutation (Figures 4A and 4B) were degraded slowly. Degradation of the arrested fragment will be necessary to avoid permanent jamming of the translation apparatus by the elongation-arrested product. In this respect, it is conceivable that the ssrAmediated trans-translation system (Karzai et al., 2000) is involved in the degradation of the arrested fragment. It is conceivable that the secM messenger RNA (McNicholas et al., 1997) contains a region where ribosomes tend to stall (Wolin and Walter, 1988). The electrophoretic mobility of the arrested fragment suggests that the translation pause occurs at a point close to the end of the secM coding region. McNicholas et al. (1997) proposed two stem–loop structures in the secM-secA messenger RNA, one (helix I) around the 3⬘ end of secM, involved in the secretion response, and the other (helix II) in the intergenic-secA region, involved in the SecA translation initiation and its autogenous repression. Our results nicely match their proposal. When a ribosome stalls in this region, these helices may be disrupted for extended lengths of time, with concomitant exposure of the secA Shine-Dalgarno sequence that is otherwise occluded in helix II (Oliver et al., 1998). As the secM plasmids used in this study did not include the complete helix II sequence, the helix I region (secretion responsive element; McNicholas et al., 1997) seems to be sufficient to trigger the translation halt. The secM translation pause is transient in the presence of active translocation and prolonged in the absence of translocation. Since SecM signal sequence mutations cause the prolongation of the arrest in otherwise wild-type cells, it is the translocation status of nascent SecM itself, but not the general cellular secretion activity, that determines the extent of translation pause. Although our finding of SecM translation arrest was originally based on the observations made on the cloned SecM, we substantiated that the chromosomally en-

coded SecM also undergoes secretion defect–induced arrest, which was well correlated with the increase in SecA translation. What mechanism might couple the secretion status of SecM and its translation arrest? We have shown that incorporation of azetidine into the nascent SecM leads to an alleviation of the elongation arrest under the SecYdefective conditions, while the secY24/Syd translocation defect was not suppressed by azetidine. This amino acid analog might have slowed down the peptide chain elongation to some extent. If the elements in the messenger RNA alone had determined the translation arrest, a slowed translation should have favored the arrest. The experimental result that azetidine actually acted to cancel the arrest argues against the above notion. Instead, the azetidine effect is consistent with the notion that the nascent SecM has a positive role in the establishment of the prolonged arrest. Without translocation, the nascent SecM product may be cotranslationally folded in the cytosol into a domain that might be called an “arrestase,” which interacts with the ribosome to establish a prolonged translation arrest. The stalled ribosome can in turn interfere with the secondary structure formation of the secM-secA messenger RNA (McNicholas et al., 1997), resulting in an increased initiation frequency of secA translation. In contrast, under the normal translocation conditions, nascent SecM may undergo cotranslational translocation, leaving no opportunity for it to fold in the cytosol into the active arrestase domain. Alternatively, or in addition, the SecM translocation may be initiated at later stages of translation and may act to cancel any cotranslational folding as well as any arrestase activity that a folded domain is going to attain. It should be noted that the arrestase activity of the nascent SecM chain is still hypothetical and that the possibility is open that other factor(s) also participate in the establishment of the translation arrest. We found that translocation of SecM across the membrane depended on SRP, SecA, and SecY but not on SecB (data not shown for the SecB independence). Recent studies revealed that targeting of membrane proteins requires SRP (Ulbrandt et al., 1997; Tian et al., 2000), although roles played by SecA in the SRP-targeted translocation is unclear (Valent et al., 1998; Koch et al., 1999). Our results show that SecM uses both SRP and SecA. Recently, Sarker et al. (2000) corrected the secM initiation codon; it is a GUG codon located at 69 nucleotides upstream of the originally assigned start codon. Thus, the N-terminal hydrophilic region, as well as the hydrophobic core of the SecM signal sequence, is longer than those of average signal sequences (Sarker et al., 2000). These peculiar features of SecM signal peptide may make it to be recognized by SRP for cotranslational targeting. Thus, SecM is endowed with the ability of monitoring cellular activities of both protein export, as its export is Sec dependent, and membrane protein integration, as its targeting is SRP dependent. The present work revealed a novel localization-directed regulatory mechanism, in which localization of SecM, as it is translated, determines its own elongation arrest as well as the expression of the cis-located downstream secA gene.

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Experimental Procedures Plasmid Constructions A secM region was cloned by amplifying it from the chromosome of E. coli strain MC4100 (Silhavy et al., 1984) using a pair of primers, 5⬘-GAATTCGAGCTCGGCAATAACGTGAGTGG-3⬘ and 5⬘-AAGCTTGC ATGCATAATAAAATCTCAAACG-3⬘, and cloning it into SacI-SphIdigested pUC118. The resulting plasmid was named pNH21. Plasmid pNH26 had the SacI-SphI secM fragment of pNH21 cloned into pSTV28, a pACYC184-based lac promoter vector from Takara. pNH22 encoding SecM-Met6 was constructed similarly to pNH21, except that the downstream primer was 5⬘-AAGCTTGCATGCTTACA TCATCATCATCATCATGGTGAGGCGTTGAGGACCGCC-3⬘. pNH5 was a pSTV28 version of secM-met6 plasmid. pNH1, was a pBluescriptKS(-) derivative having the SacI-HindIII fragment from pNH22. pNH32, encoding SecM-HA-Met6, was constructed by inserting 5⬘-TACCCATACGATGTTCCTGACTATGCG-3⬘ (for influenza virus hemagglutinin epitope sequence) between the SecM and Met6 coding sequences of pNH22, using QuickChange mutagenesis kit (Stratagene). Similarly, pNH31 encoded SS-HA-SecM-Met6, in which HA was inserted between Ala-46 and Thr-47 of SecM-Met6. In this work, the initiation GUG codon (Sarker et al., 2000) is adopted as the first codon of secM, and amino acid residues are numbered accordingly. We assume that the mature sequence starts at Ala-38. pNH30 encoding ⌬LGLPA-SecM-Met6 was constructed by introducing the deletion for Leu-29-Ala-33 into pNH22 by site-directed mutagenesis. pNH7 encoding ⌬SS-’SecM-Met6 was constructed as follows. A plasmid pNH10 was first constructed, in which the first two codons (ATGACC) of lacZ␣ on pUC118 were replaced by the NsiI recognition sequence (ATGCAT). A DNA fragment for Arg-49 to the C terminus of SecM-Met6 was amplified from pNH22 and cloned into NsiI-SphIdigested pNH10. All the constructions were confirmed for their secM region sequences. Pulse–Chase Analysis of SecM Biosynthesis E. coli strain GN40 (Matsumoto et al., 1997) harboring pSTD343, a pACYC184-based plasmid carrying lacI (Y. Akiyama, personal communication), was used as a sec⫹ “wild-type” strain, into which the second plasmid expressing a SecM derivative was introduced. Cells were grown at 37⬚C in minimal medium M9 (Silhavy et al., 1984) supplemented with 18 amino acids (20 ␮g/ml each except Met and Cys), 0.4% glucose, 2 ␮g/ml thiamine, and appropriate antibiotics to maintain the plasmids. Cells in an exponential phase were induced for SecM expression with 1 mM isopropyl-1-thio-␤-D-galactoside (IPTG) and 5 mM cyclic AMP, typically for 30 min, and pulse labeled with [35S]methionine for 1 min. Chase was then initiated by adding unlabeled L-methionine (200 ␮g/ml). At each time point indicated, a portion of culture was treated with trichloroacetic acid, and denatured total cell proteins were collected, washed, and dissolved in SDS (Matsumoto et al., 1997). Samples were then processed for immunoprecipitation (Matsumoto et al., 1997) using a mixture of rabbit polyclonal antisera against synthetic peptides with Glu-39-Ser-54 as well as Thr-127-Ala-142 sequences of SecM. Immunoprecipitates were subjected to SDS-PAGE and phosphor imager (BAS1800, Fuji Film) visualization of the labeled proteins. CTABr Fractionation To precipitate nucleic acids, 500 ␮l of 2% (w/v) CTABr and 500 ␮l of 0.5 M sodium acetate (pH 4.7) were added to 50 ␮l of SDSsolubilized extracts from radio-labeled cells prepared as above. After standing on ice for 10 min, samples were incubated further at 30⬚C for 10 min and centrifuged at 15,000 rpm for 15 min at room temperature in a microfuge. Precipitates were washed twice with 500 ␮l of acetone-HCl (19:1) and dissolved in 50 mM Tris-HCl (pH 8.1) containing 1% SDS and 1 mM EDTA for immunoprecipitation and SDS-PAGE as described above. The supernatant was treated with 20% trichloroacetic acid to recover protein contents and analyzed similarly. Acknowledgments We thank Ei-ichi Matsuo for his advice on in vitro transcriptiontranslation experiments as well as for generously supplying the re-

agents required; T. Silhavy, J. Luirink, H. Hara, and Y. Akiyama for bacterial strains and plasmids; and Yoshinori Akiyama and Hiroyuki Mori for helpful discussion. Thanks are also due to Don Oliver for the information about the secM translation initiation site and for discussion regarding the nomenclature of this gene. This work was supported by CREST, Japan Science and Technology Corporation, and by grants from the Ministry of Education, Science, and Culture, Japan. Received August 7, 2000; revised November 6, 2000. References Baba, T., Jacq, A., Brickman, E., Beckwith, J., Taura, T., Ueguchi, C., Akiyama, Y., and Ito, K. (1990). Characterization of cold-sensitive secY mutants of Escherichia coli. J. Bacteriol. 172, 7005–7010. Duong, F., Eichler, J., Price, A., Leonard, M.R., and Wickner, W. (1997). Biogenesis of the gram-negative bacterial envelope. Cell 91, 567–573. Economou, A. (1998). Bacterial preprotein translocase: mechanism and conformational dynamics of a processive enzyme. Mol. Microbiol. 27, 511–518. Fekkes, P., and Driessen, A.J. (1999). Protein targeting to the bacterial cytoplasmic membrane. Microbiol. Mol. Biol. Rev. 63, 161–173. Fikes, J.D., and Bassford, P.J. (1989). Novel secA alleles improve export of maltose-binding protein synthesized with a defective signal peptide. J. Bacteriol. 171, 402–409. Gilmore, R., Collins, P., Johnson, J., Kellaris, K., and Rapiejko, P. (1991). Transcription of full-length and truncated mRNA transcripts to study protein translocation across the endoplasmic reticulum. Methods Cell Biol. 34, 223–239. Hara, H., Abe, N., Nakakouji, M., Nishimura, Y., and Horiuchi, K. (1996). Overproduction of penicillin-binding protein 7 suppresses thermosensitive growth defect at low osmolarity due to an spr mutation of Escherichia coli. Microb. Drug Resist. 2, 63–72. Karzai, A.W., Roche, E.D., and Sauer, R.T. (2000). The SsrA-SmpB system for protein tagging, directed degradation and ribosome rescue. Nat. Struct. Biol. 7, 449–455. Keiler, K.C., Waller, P.R., and Sauer, R.T. (1996). Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271, 990–993. Koch, H.G., Hengelage, T., Neumann-Haefelin, C., MacFarlane, J., Hoffschulte, H.K., Schimz, K.L., Mechler, B., and Mu¨ller, M. (1999). In vitro studies with purified components reveal signal recognition particle (SRP) and SecA/SecB as constituents of two independent protein-targeting pathways of Escherichia coli. Mol. Biol. Cell 10, 2163–2173. Kusters, R., Lentzen, G., Eppens, E., van Geel, A., van der Weijden, C.C., Wintermeyer, W., and Luirink, J. (1995). The functioning of the SRP receptor FtsY in protein-targeting in E. coli is correlated with its ability to bind and hydrolyse GTP. FEBS Lett. 372, 253–258. Luirink, J., ten Hagen-Jongman, C.M., van der Weijden, C.C., Oudega, B., High, S., Dobberstein, B., and Kusters, R. (1994). An alternative protein targeting pathway in Escherichia coli: studies on the role of FtsY. EMBO J. 13, 2289–2296. Matsumoto, G., Yoshihisa, T., and Ito, K. (1997). SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane. EMBO J. 16, 6384–6393. Matsuo, E., Mori, H., Shimoike, T., and Ito, K. (1998). Syd, a SecYinteracting protein, excludes SecA from the SecYE complex with an altered SecY24 subunit. J. Biol. Chem. 273, 18835–18840. McNicholas, P., Salavati, R., and Oliver, D. (1997). Dual regulation of Escherichia coli secA translation by distinct upstream elements. J. Mol. Biol. 265, 128–141. Nishiyama, K., Hanada, M., and Tokuda, H. (1994). Disruption of the gene encoding p12 (SecG) reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli at low temperature. EMBO J. 13, 3272–3277. Oliver, D., Norman, J., and Sarker, S. (1998). Regulation of Escherichia coli secA by cellular protein secretion proficiency requires an

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