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• W2035830103 abstract "Syd is an Escherichia coli cytosolic protein that interacts with SecY. Overproduction of this protein causes a number of protein translocation-related phenotypes, including the strong toxicity against the secY24 mutant cells. Previously, this mutation was shown to impair the interaction between SecY and SecE, the two fundamental subunits of the membrane-embedded part of protein translocase. We have now studied in vitrothe mechanisms of the Syd-directed inhibition of protein translocation. Pro-OmpA translocation into inverted membrane vesicles (IMVs) prepared from the secY24 mutant cells as well as the accompanied translocation ATPase activity of SecA were rapidly inhibited by purified Syd protein. In the course of protein translocation, high affinity binding of preprotein-bearing SecA to the translocase on the IMV is followed by ATP-driven insertion of the 30-kDa SecA segment into the membrane. Our experiments using 125I-labeled SecA and the secY24 mutant IMV showed that Syd abolished both the high affinity SecA binding and the SecA insertion. Syd was even able to release the inserted form of SecA that had been stabilized by a nonhydrolyzable ATP analog. Syd affected markedly the proteolytic digestion pattern of the IMV-integrated SecY24 protein, suggesting that Syd exerts its inhibitory effect by interacting directly with the SecY24 protein. In accordance with this notion, a SecY24 variant with a second site mutation (secY249) resisted the Syd action bothin vivo and in vitro. Thus, Syd acts against the SecY24 form of translocase, in which SecY-SecE interaction has been compromised, to exclude the SecA motor protein from the SecYE channel complex. Syd is an Escherichia coli cytosolic protein that interacts with SecY. Overproduction of this protein causes a number of protein translocation-related phenotypes, including the strong toxicity against the secY24 mutant cells. Previously, this mutation was shown to impair the interaction between SecY and SecE, the two fundamental subunits of the membrane-embedded part of protein translocase. We have now studied in vitrothe mechanisms of the Syd-directed inhibition of protein translocation. Pro-OmpA translocation into inverted membrane vesicles (IMVs) prepared from the secY24 mutant cells as well as the accompanied translocation ATPase activity of SecA were rapidly inhibited by purified Syd protein. In the course of protein translocation, high affinity binding of preprotein-bearing SecA to the translocase on the IMV is followed by ATP-driven insertion of the 30-kDa SecA segment into the membrane. Our experiments using 125I-labeled SecA and the secY24 mutant IMV showed that Syd abolished both the high affinity SecA binding and the SecA insertion. Syd was even able to release the inserted form of SecA that had been stabilized by a nonhydrolyzable ATP analog. Syd affected markedly the proteolytic digestion pattern of the IMV-integrated SecY24 protein, suggesting that Syd exerts its inhibitory effect by interacting directly with the SecY24 protein. In accordance with this notion, a SecY24 variant with a second site mutation (secY249) resisted the Syd action bothin vivo and in vitro. Thus, Syd acts against the SecY24 form of translocase, in which SecY-SecE interaction has been compromised, to exclude the SecA motor protein from the SecYE channel complex. Protein translocation across the E. coli plasma membrane is facilitated by multiple Sec factors (for reviews, see Refs. 1Wickner W. Leonard M.R. J. Biol. Chem. 1996; 271: 29514-29516Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar and 2Ito K. Genes Cells. 1996; 1: 337-346Crossref PubMed Scopus (40) Google Scholar). A membrane-interacting ATPase, SecA, binds with high affinity to the membrane containing the functional SecY/SecE components (3Hartl F.-U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (447) Google Scholar). This is then followed by the ATP-dependent insertion of a SecA segment into the membrane (4Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (482) Google Scholar). ATP hydrolysis-dependent release of the preprotein and deinsertion of SecA allow the next cycle of polypeptide movement (4Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (482) Google Scholar). SecY, SecE, and SecG are the essential core components in the membrane, having 10, 3, and 2 transmembrane segments, respectively. They not only provide the high affinity binding site for SecA (3Hartl F.-U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (447) Google Scholar) but allow productive SecA insertion (4Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (482) Google Scholar, 5Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar), and they are thought to provide a channel-like pathway for the movement of polypeptide chain. These membrane proteins form a trimeric complex (6Brundage L. Hendrick J.P. Schiebel E. Driessen A.J.M. Wickner W. Cell. 1990; 62: 649-657Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 7Joly J.C. Leonard M.R. Wickner W.T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4703-4707Crossref PubMed Scopus (56) Google Scholar, 8Brundage L. Fimmel C.J. Mizushima S. Wickner W. J. Biol. Chem. 1992; 267: 4166-4170Abstract Full Text PDF PubMed Google Scholar) in which SecY interacts independently with SecE and SecG (9Homma T. Yoshihisa T. Ito K. FEBS Lett. 1997; 408: 11-15Crossref PubMed Scopus (26) Google Scholar). Proteoliposomes reconstituted only from SecY and SecE exhibit some basic translocation activities in conjunction with SecA (10Akimaru J. Matsuyama S. Tokuda H. Mizushima S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6545-6549Crossref PubMed Scopus (171) Google Scholar). In addition, the inclusion of SecG markedly stimulates the translocation activities of the SecYE proteoliposomes (8Brundage L. Fimmel C.J. Mizushima S. Wickner W. J. Biol. Chem. 1992; 267: 4166-4170Abstract Full Text PDF PubMed Google Scholar, 11Nishiyama K. Mizushima S. Tokuda H. EMBO J. 1993; 12: 3409-3415Crossref PubMed Scopus (130) Google Scholar). In vivo, SecY requires SecE as the stabilizing partner (12Matsuyama S. Akimaru J. Mizushima S. FEBS Lett. 1990; 269: 96-100Crossref PubMed Scopus (92) Google Scholar,13Taura T. Baba T. Akiyama Y. Ito K. J. Bacteriol. 1993; 175: 7771-7775Crossref PubMed Scopus (68) Google Scholar). Thus, uncomplexed forms of SecY are degraded rapidly by the FtsH proteolytic system (14Kihara A. Akiyama Y. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4532-4536Crossref PubMed Scopus (210) Google Scholar). We proposed previously that a dominant negative variant of SecY, SecY−d1, with a small internal deletion, sequesters SecE in an inactive complex (15Shimoike T. Akiyama Y. Baba T. Taura T. Ito K. Mol. Microbiol. 1992; 6: 1205-1210Crossref PubMed Scopus (28) Google Scholar). Intragenic suppressor mutations that abolish the dominant effect preferentially occur within or around cytoplasmic domain 4 (C4) of SecY (16Baba T. Taura T. Shimoike T. Akiyama Y. Yoshihisa T. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4539-4543Crossref PubMed Scopus (52) Google Scholar). ThesecY24 amino acid substitution (Gly240 → Asp) in C4 (17Shiba K. Ito K. Yura T. Cerretti D.P. EMBO J. 1984; 3: 631-635Crossref PubMed Scopus (121) Google Scholar), which also suppressessecY−d 1 intragenically, abolishes the SecY-SecE affinity co-isolation (9Homma T. Yoshihisa T. Ito K. FEBS Lett. 1997; 408: 11-15Crossref PubMed Scopus (26) Google Scholar, 16Baba T. Taura T. Shimoike T. Akiyama Y. Yoshihisa T. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4539-4543Crossref PubMed Scopus (52) Google Scholar). Thus, C4 is an essential cytoplasmic domain that is important for the interaction between SecY and SecE. The importance of the second cytoplasmic segment (C2) of SecE for SecY-SecE interaction was also shown (18Pohlschröder M. Murphy C. Beckwith J. J. Biol. Chem. 1996; 271: 19908-19914Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The secY−d 1 mutation allowed us to identify a new gene, syd, as a multicopy suppressor against this mutation (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The syd gene product (Syd) is a hydrophilic protein of 181 amino acid residues, which is associated weakly with the membrane, presumably via its interaction with the SecY protein. When Syd is overproduced in SecY−d1-expressing cells, it preferentially stabilizes the chromosomally encoded wild-type SecY protein, which is now given a greater opportunity to associate with SecE. This is the proposed suppression mechanism. Another striking phenotype associated with overproduction of Syd is its strong toxicity against the secY24 mutant cells. Presumably because of the defect in SecY-SecE interaction, the SecY24 mutant protein is degraded at 42 °C. This degradation is primarily responsible for the protein export defect (14Kihara A. Akiyama Y. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4532-4536Crossref PubMed Scopus (210) Google Scholar). However, the Syd-mediated impairment of protein export and cell viability is not accompanied by degradation of the SecY protein (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Thus, Syd seems to directly interfere with the functioning of the SecY24 mutant protein. These observations indicate that Syd provides a useful means to investigate into the nature of protein interactions involving SecY. Through our reversion studies of the secY24 mutant with respect to its susceptibility to Syd overproduction, we identified three new alleles of secY (20Matsuo E. Ito K. Mol. Gen. Genet. 1998; 258: 240-249Crossref PubMed Scopus (9) Google Scholar). While two of them affected residue 240 (the residue altered by secY24) of SecY, the third mutation, termed secY249 (for Ala249 → Val) alleviated the Syd sensitivity of the secY24 alteration (when placed in cis configuration with secY24) without restoring the temperature resistance or the SecY-SecE interaction. Thus, the secY249 mutation may affect the interaction between SecY and Syd. This work was focused on the ability of Syd to inhibit protein translocase when its SecY subunit is altered to the SecY24 mutant form. Our in vitro experiments show that Syd interferes with productive interaction between SecA and the membrane-integrated SecYE complex. Plasmid pST67 contained the syd gene placed under the lacpromoter and a translation-initiating SD sequence (5′-AAGGAG-3′) and was used as a Syd-overproducing plasmid. For its construction, a polymerase chain reaction was programmed on pST30 (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) with primers 5′-GGGGTACCCCAAGGAGAACAGCTATGGACGATTTGACCGC-3′ and 5′-GCCCTAGGGCAATGTCTTCTCCGAATT-3′ to produce a 0.57-kilobase pair fragment, which was then treated with KpnI andBamHI and cloned into pUC18. The resulting plasmid was confirmed for the Syd-coding region by sequencing. E. colistrain AD202 was a derivative of MC4100 (21Casadaban M.J. J. Mol. Biol. 1976; 104: 541-555Crossref PubMed Scopus (1298) Google Scholar) carryingompT::kan (22Akiyama Y. Ito K. Biochem. Biophys. Res. Commun. 1990; 167: 711-715Crossref PubMed Scopus (118) Google Scholar). EM147 was a derivative of MC4100 carrying syd::kan (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). EM166 was a derivative of MC4100 carryingsyd::cat and constructed essentially as described previously (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), except that it carried the chloramphenicol resistance determinant (cat) (instead of kanpreviously used) in the middle of the syd gene. Mutants carrying the secY24 or secY24–249 mutation were described previously (20Matsuo E. Ito K. Mol. Gen. Genet. 1998; 258: 240-249Crossref PubMed Scopus (9) Google Scholar). These mutations were transduced into the genetic background of strain TW155 (a ΔuncB uncCderivative of AD202; Ref. 5Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar) and named EM153 and EM152, respectively. For these constructions, zhd-33::Tn10(17Shiba K. Ito K. Yura T. Cerretti D.P. EMBO J. 1984; 3: 631-635Crossref PubMed Scopus (121) Google Scholar) was used as a selective marker, and transductants were examined for temperature-sensitivity and Syd sensitivity (20Matsuo E. Ito K. Mol. Gen. Genet. 1998; 258: 240-249Crossref PubMed Scopus (9) Google Scholar); an isogenicsecY+ strain was named EM154. Their further derivatives that received syd::cat from EM166 were named EM168 (secY24 syd::cat), EM167 (secY24–249 syd::cat), and EM169 (secY+ syd::cat). IMVs 1The abbreviations used are: IMV, inverted inner membrane vesicle; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; PAGE, polyacrylamide gel electrophoresis. from strains of a particular secY phenotype exhibited essentially indistinguishable activities whether they were from strains of thesyd+ or thesyd::cat background. Syd-overproducing cells (EM147/pST67 or CU141 (23Akiyama Y. Ogura T. Ito K. J. Biol. Chem. 1994; 269: 5218-5224Abstract Full Text PDF PubMed Google Scholar)/pST67) were grown at 37 °C to a middle to late log phase in 1.5 liters of peptone medium (20Matsuo E. Ito K. Mol. Gen. Genet. 1998; 258: 240-249Crossref PubMed Scopus (9) Google Scholar) supplemented with 0.017m KH2PO4, 0.072 mK2HPO4, and ampicillin (50 μg/ml). Isopropyl β-d-thiogalactopyranoside (1 mm) was added to induce the lac promoter-directed overexpression ofsyd. Syd was purified essentially as described previously (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), desalted by a NAP-25 column (Amersham Pharmacia Biotech) equilibrated with 50 mm Tris-HCl (pH 7.5) and 10% glycerol and stored at −80 °C. Protein concentration was determined by a Micro BCA protein assay kit (purchased from Pierce) using bovine serum albumin as a standard. IMVs were prepared as described previously (24Yoshihisa T. Ito K. J. Biol. Chem. 1996; 271: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) from cells of the specified strains that had been grown at 30 °C. They were treated with 6 m urea for 1 h at 0 °C (25Taura T. Yoshihisa T. Ito K. Biochimie. 1997; 79: 517-521Crossref PubMed Scopus (30) Google Scholar) when specified. Pro-OmpA was synthesizedin vitro in the presence of [35S]methionine as described previously (26Baba T. Jacq A. Brickman E. Beckwith J. Taura T. Ueguchi C. Akiyama Y. Ito K. J. Bacteriol. 1990; 172: 7005-7010Crossref PubMed Google Scholar). The transcription-translation mixture was then mixed with chloramphenicol (100 μg/ml), sodium succinate (5 mm), IMV (100 μg of protein/ml), and SecA (40 μg/ml). Various concentrations of Syd were added before SecA when specified. After incubation at 37 °C, samples were placed on ice and treated with proteinase K (100 μg/ml) at 0 °C for 30 min. Reactions were stopped by the addition of 1 mm phenylmethylsulfonyl fluoride followed by trichloroacetic acid precipitation. Samples were separated by SDS-PAGE and visualized by autoradiography as described previously (26Baba T. Jacq A. Brickman E. Beckwith J. Taura T. Ueguchi C. Akiyama Y. Ito K. J. Bacteriol. 1990; 172: 7005-7010Crossref PubMed Google Scholar), and the bands were quantified by a Fuji BAS2000 phosphor imager. The reaction mixture (50 μl) contained urea-washed IMV (100 μg/ml), SecA (40 μg/ml) and ATP (4 mm) in buffer composed of 50 mm Tris-HCl (pH 7.2), 30 mm KCl, 30 mm NH4Cl, 2.5 mm magnesium acetate, and 0.5 mg/ml bovine serum albumin (fatty acid-free, from Nacalai Tesque). It was further supplemented with pro-3His-OmpA′ (66 μg/ml; Ref. 24Yoshihisa T. Ito K. J. Biol. Chem. 1996; 271: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and/or Syd (100 μg/ml) as specified. After incubation at 37 °C, inorganic phosphate liberated was quantified (5Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar, 27Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (463) Google Scholar). To obtain translocation ATPase activities, values in the absence of pro-3His-OmpA′ were subtracted from those in its presence. SecA was iodinated with125I and subjected to the insertion assay essentially as described previously (4Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (482) Google Scholar, 5Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar), except that Syd was added when indicated. SecA binding assays were similarly performed according to previously published procedures (3Hartl F.-U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (447) Google Scholar, 5Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar). Urea-treated IMVs were incubated in the presence or absence of Syd (100 μg/ml) with various concentrations of trypsin at 0 °C for 20 min. A purified preparation of pro-OmpA (26 μg/ml) (5Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar, 28Sato K. Mori H. Yoshida M. Tagaya M. Mizushima S. J. Biol. Chem. 1997; 272: 20082-20087Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) was added in some experiments, but it did not affect the digestion patterns. Reactions were stopped by the addition of 1 mm phenylmethylsulfonyl fluoride followed by trichloroacetic acid precipitation. The precipitates were washed with acetone, solubilized in SDS sample buffer at 37 °C for 5 min, and separated by SDS-PAGE (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). SecY and SecE were then visualized by immunoblotting (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), using antiserum against the N-terminal 22 residues of SecY (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and antiserum against residues 64–81 of SecE, respectively. The latter antiserum, a custom product of Sawady Technology, was raised in a rabbit using the synthetic peptide conjugated with keyhole limpet hemocyanin. Kaleidoscope prestained standards (purchased from Bio-Rad) were used as molecular weight markers in electrophoresis. Overproduction of Syd in the secY24 mutant cells leads to the severest extent of inhibition of protein export known for E. coli sec mutants (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and appeared to merit investigations in vitro. We prepared IMVs from wild-type and secY24 mutant cells grown at 30 °C, the permissive temperature for the mutant. An in vitro translation product of pro-OmpA, labeled with [35S]methionine, was subjected to posttranslational translocation into IMV. When IMVs from secY24 mutant cells were preincubated at 0 °C with increasing concentrations of Syd, translocation of pro-OmpA was progressively inhibited (Fig. 1). In contrast, pro-OmpA translocation into the wild-type IMV was not appreciably affected by Syd of up to 100 μg/ml (Fig. 1). Dose-dependent inhibition of translocation was also observed when a crude cytosol fraction from the Syd-overproducing cells was added to the reaction with the SecY24 IMV. Immunoblotting experiments showed that the concentrations of Syd protein required for a particular level of inhibition were similar between the purified sample and the crude cytosolic fraction, indicating that Syd had not been inactivated during the purification procedures (data not shown). Neither bovine serum albumin, up to 1 mg/ml, nor heat-treated Syd exerted any appreciable inhibitory effect (data not shown). Thus, this inhibition was due to the functional Syd protein and not to some nonspecific factors such as increased protein concentration. When the SecY24 IMV was first incubated with excess Syd, sedimented, and subjected to the translocation assay, substantial levels of translocation of pro-OmpA were observed. Syd concentration in the second incubation mixture was about 15% of that before centrifugation. We suggest that Syd inhibits translocation while in an equilibrium of association and dissociation with IMV. In fact, preincubation was not necessary, since Syd added after the initiation of translocation reaction also prevented the subsequent increase of the translocated molecules (data not shown). None of the translocase subunits was proteolyzed during the pretreatment with Syd or after the incubation at 37 °C with Syd, as confirmed by direct immunoblotting experiments (data not shown). The ATP hydrolysis activity of SecA that is stimulated by a precursor protein and membrane vesicles with intact SecYEG complex is termed translocation ATPase (29Lill R. Cunningham K. Brundage L.A. Ito K. Oliver D. Wickner W. EMBO J. 1989; 8: 961-966Crossref PubMed Scopus (309) Google Scholar). We found that Syd was a potent inhibitor of translocation ATPase when SecA was combined with the SecY24 IMV. Whereas Syd (100 μg/ml) inhibited this activity almost completely (Fig. 2,solid triangles), the same concentration of Syd only negligibly affected the activity in combination with the wild-type IMV (Fig. 2, solid circles). The inhibitory effect of Syd was specific for ATP hydrolysis that was coupled with translocation, such that neither the membrane ATPase activity (in the absence of preprotein) nor the intrinsic ATPase activity (without any other macromolecules) was affected significantly (data not shown). The addition of Syd into the reaction mixture in which SecA translocation ATPase was already activated by pro-OmpA and SecY24 IMV resulted in almost instantaneous inhibition of the ongoing reaction (Fig. 3).Figure 3Effects of Syd on ongoing reactions of SecA translocation ATPase. Two reactions of SecA translocation ATPase using IMVs from the secY24 mutant (EM168) were initiated at 37 °C. After 5 min, Syd (100 μg/ml) was added to one reaction (solid triangles), whereas the other reaction received the same volume of the buffer (open triangles).View Large Image Figure ViewerDownload (PPT) We examined the SecA insertion reaction (4Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (482) Google Scholar) using125I-labeled SecA and urea-washed wild-type and SecY24 IMVs. In the absence of added Syd, both wild-type IMV and SecY24 IMV supported the pro-OmpA- and ATP-dependent formation of a membrane-protected 30-kDa fragment (Fig. 4, lane 4), although the efficiency was slightly lower for the SecY24 IMV. A nonhydrolyzable ATP analog, AMP-PNP, supported apparently pro-OmpA-independent insertion of SecA (Fig. 4, lane 6) as reported previously (5Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar, 30Economou A. Pogliano J.A. Beckwith J. Oliver D.B. Wickner W. Cell. 1995; 83: 1171-1181Abstract Full Text PDF PubMed Scopus (271) Google Scholar). When Syd was included in these reactions, the SecY24 IMV did not support any detectable insertion of SecA, irrespective of whether ATP or AMP-PNP was used (Fig. 4, middle panel, lanes 1–3). In contrast, Syd inhibited SecA insertion into the wild-type IMV only slightly (Fig. 4, upper panel, lanes 1–3). These results indicate that Syd uncouples the SecA functions from the SecY24-mediated protein translocation reaction. To define the target reaction of the Syd inhibition, we carried out SecA binding assays. Various concentrations of SecA and IMV were incubated at 0 °C, and the membrane-bound SecA was isolated for the Scatchard analysis of the binding. SecA binding to the SecY24 IMV was found to be abolished almost completely by Syd. Thus, the high affinity phase of the binding was hardly observed in the presence of Syd (Fig. 5 B, solid triangles). High affinity binding of SecA to the wild-type IMV was lowered by Syd to only a small extent (Fig. 5 A, compare open and solid circles). These results suggest that the Syd action to the SecY24 IMV can essentially be ascribed to the elimination of the high affinity SecA binding. We then examined whether Syd interferes with the SecA-SecY interaction passively, for instance by steric hindrance, or if it acts more actively, for instance to change the subunit arrangement of the SecY-SecE complex. For this purpose, we studied the effect of Syd on a state of SecA that had already been inserted and stabilized as such by AMP-PNP. 125I-Labeled SecA was first allowed to insert into wild-type or SecY24 IMV in the presence of AMP-PNP and then subjected to the second incubation with either Syd or buffer alone. The second incubation in the presence of Syd resulted in the disappearance of the 30-kDa fragment inserted into the SecY24 IMV (Fig. 6, lower panel,lane 1), whereas SecA inserted into the wild-type IMV persisted (Fig. 6, upper panel,lane 1). Incubation without Syd did not cause any change in the intensity of the inserted SecA (compare Fig. 4,lane 6, and Fig. 6, lane 2). Thus, Syd is able to dissociate SecA that is otherwise locked in the inserted state by the lack of ATP hydrolysis. We also noted that the AMP-PNP-stabilized SecA was partially released from the SecY24 IMV, but not from the wild-type IMV, when unlabeled excess SecA was added, suggesting that SecA insertion occurs somewhat weakly against the mutant IMV. ThesecY24 alteration in SecY weakens the interaction between SecY and SecE such that the SecY24-SecE complex comes apart after solubilization with a nonionic detergent (9Homma T. Yoshihisa T. Ito K. FEBS Lett. 1997; 408: 11-15Crossref PubMed Scopus (26) Google Scholar, 26Baba T. Jacq A. Brickman E. Beckwith J. Taura T. Ueguchi C. Akiyama Y. Ito K. J. Bacteriol. 1990; 172: 7005-7010Crossref PubMed Google Scholar). We examined trypsin digestion patterns of SecY and SecE in the membrane-integrated state. Wild-type and SecY24 IMV were treated with increasing concentrations of trypsin, and SecY and SecE were detected by immunoblotting using antisera against the N-terminal sequence of SecY and the central cytoplasmic region of SecE, respectively. Treatment of the wild-type IMV with low concentrations of trypsin produced an N-terminal fragment of about 21 kDa (Fig. 7, lanes 17 and 18). Its apparent molecular size suggests that the site of this cleavage was within cytoplasmic region 4 (C4). The SecY24 IMV gave different trypsin digestion patterns in two respects. First, the overall trypsin sensitivity as assessed by the disappearance of the intact SecY molecules was significantly higher for the SecY24 IMV than the wild-type IMV (Fig. 7, compare lanes 6–10 with lanes 16–20). Second, the 21-kDa N-terminal fragment produced from the SecY24 IMV (Fig. 6,lane 7) was further shortened to a slightly faster migrating product when trypsin concentrations were increased (Fig. 7, lanes 8 and 9). Although the SecY24 amino acid alteration (Gly240 to Asp) is also in C4 domain of SecY, it is unrelated to any amino acid residues directly relevant to the trypsin-mediated hydrolysis of peptide bonds. Thus, this sequence alteration may have caused a change in local conformation of SecY or in subunit arrangements of the SecYEG complex. We found a striking difference in the trypsin susceptibility of SecE between the wild-type IMV and the SecY24 IMV. SecE in the former IMV resisted trypsin of the highest concentration examined (Fig. 7,lower panel, lanes 16–20), although some decrease in the intensity of SecE, in parallel with that of the intact SecY, was observed at higher trypsin concentrations (Fig. 7, lanes 11–20, compare upper andlower panels). In contrast, SecE in the SecY24 IMV was digested almost completely by trypsin (Fig. 7, lower panel, lanes 9 and 10). SecE was more trypsin-sensitive than SecY24 itself (compareupper and lower panels forlane 5 of Fig. 7). These results lend further support to the notion that subunit arrangements of the SecYEG complex have been altered by the SecY24 amino acid alteration, resulting in significantly impaired SecY-SecE interaction. We then repeated the trypsin digestion experiments in the presence of an excess of Syd protein. While the addition of Syd did not appreciably affect the trypsin digestion profiles of SecY or SecE in the wild-type IMV, it markedly changed the pattern of SecY cleavage in the SecY24 IMV. The 21-kDa N-terminal fragment was now shifted to an apparent molecular mass of 27 kDa (Fig. 7, upper panel,lanes 3 and 4). Thus, the original two cleavage sites (presumably in C4) were somehow protected by Syd. Thus, Syd interacts specifically with SecY24. However, it did not exert any protecting effect on SecE (Fig. 7, lower panel,lanes 4 and 5). Previously, we isolated Syd-resistant variants from thesecY24 mutant (20Matsuo E. Ito K. Mol. Gen. Genet. 1998; 258: 240-249Crossref PubMed Scopus (9) Google Scholar). The mutations converged into three alleles in addition to the true reversion. Two of the alleles change residue 240 affected by the secY24 mutation, restoring the SecY-SecE interaction (20Matsuo E. Ito K. Mol. Gen. Genet. 1998; 258: 240-249Crossref PubMed Scopus (9) Google Scholar). The other allele, secY249 (for an Ala249 to Val change), is an intragenic suppressor mutation that suppresses only the sensitivity to overproduction of Syd, without any effect on the temperature-sensitive protein export defect of the secY24 mutation (20Matsuo E. Ito K. Mol. Gen. Genet. 1998; 258: 240-249Crossref PubMed Scopus (9) Google Scholar). IMVs prepared from thesecY24–249 double mutant supported pro-OmpA translocation with an efficiency similar to that of the SecY24 IMV. However, it was only slightly inhibited by Syd (Fig. 1). The double mutant IMV also supported the translocation ATPase activity of SecA even in the presence of Syd (data not shown). Insertion of the 30-kDa fragment into the double mutant IMV was not inhibited by Syd (Fig. 4,lower panel, lane 1). The SecY24–249 IMV gave tryptic digestion patterns of SecY and SecE that were essentially identical to those of the SecY24 IMV. However, they were unaffected by the presence of excess Syd (data not shown). Thus, the effects of the secY249 mutation on the phenotypes of thesecY24 mutation have been reproduced in vitro. These results suggest that the Syd effect is brought about through specific interaction between SecY and Syd, in which the residue 249 of SecY plays an essential role. Syd, originally identified as a multicopy suppressor of thesecY−d 1 mutation (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), directly interacts with SecY. Overproduction of Syd stabilizes the wild type SecY protein when it alone is overproduced or when it fails to associate with SecE because of the presence of the excess amounts of SecY−d1, the dominant negative SecY variant. The association of Syd with the plasma membrane is saturable such that SecY overproduction increases the membrane-bound state of Syd. We demonstrated in this study that Syd markedly altered the trypsin digestion pattern of SecY24 in IMV, lending further support to the notion that Syd interacts with SecY. Evidence indicates that the secY24 mutation impairs SecY-SecE interaction. The temperature sensitivity of this mutant is partially suppressible by overproduction of SecE, 2E. Matsuo, T. Taura, Y. Akiyama, and K. Ito, unpublished observations. and the mutation can act as an intragenic suppressor against the SecE-sequestering effect of the secY−d 1 mutation (16Baba T. Taura T. Shimoike T. Akiyama Y. Yoshihisa T. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4539-4543Crossref PubMed Scopus (52) Google Scholar, 20Matsuo E. Ito K. Mol. Gen. Genet. 1998; 258: 240-249Crossref PubMed Scopus (9) Google Scholar). After solubilization of the membrane with a nonionic detergent, a complex between SecY24 and SecE cannot be isolated (9Homma T. Yoshihisa T. Ito K. FEBS Lett. 1997; 408: 11-15Crossref PubMed Scopus (26) Google Scholar, 16Baba T. Taura T. Shimoike T. Akiyama Y. Yoshihisa T. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4539-4543Crossref PubMed Scopus (52) Google Scholar). The temperature-sensitive protein export defect of the secY24mutant is due to the FtsH-mediated degradation of the SecY24 protein at 42 °C (14Kihara A. Akiyama Y. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4532-4536Crossref PubMed Scopus (210) Google Scholar, 16Baba T. Taura T. Shimoike T. Akiyama Y. Yoshihisa T. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4539-4543Crossref PubMed Scopus (52) Google Scholar). These observations suggest that the impaired SecY-SecE interaction is the primary cause of the secY24defect. In further support of this notion, we found that SecE in the SecY24 IMV was more accessible to trypsin digestion than that in the wild-type IMV. The temperature sensitivity of the secY24mutant could be due to temperature-dependent exacerbation of the interaction defect, temperature-dependent activation of the proteolytic system, or both. When Syd is overproduced in the secY24 mutant, protein export is rapidly blocked followed by the loss of viability even at 30 °C (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The severity of this block makes it the most useful system to accumulate chemical amounts of a precursor protein with uncleaved signal sequence within the cell (24Yoshihisa T. Ito K. J. Biol. Chem. 1996; 271: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The rapidity and the lack of accompanied SecY proteolysis suggest that this inhibition is brought about by direct interference by Syd with the translocase functions. We reproduced the inhibitory effect of Syd in the in vitrotranslocation reaction of pro-OmpA. The concentration of Syd required for 50% inhibition of translocation was 5–10 μg/ml (or 250–500 nm) in a reaction containing IMV of 100 μg/ml proteins. Assuming that a cell contains 500 molecules of SecY (31Matsuyama S. Fujita Y. Sagara K. Mizushima S. Biochim. Biophys. Acta. 1992; 1122: 77-84Crossref PubMed Scopus (65) Google Scholar) and that 10% of total proteins are located in the inner membrane, the above concentration of IMV corresponds to 250 ng/ml (or 5 nm) of SecY. Thus, numbers of the Syd molecules required for efficient inhibition far exceed those of SecY molecules. We have shown that Syd inhibits the high affinity binding of SecA to the IMV containing the SecY24 form of the translocase subunit. This could be due to a steric hindrance by the SecY-bound Syd with respect to SecA binding. There may be a competition between Syd and SecA for the interaction with SecYEG translocase. Indeed, increased concentrations of SecA acted to partially overcome the inhibition caused by a low concentration (10 μg/ml) of Syd. Although quantitative discussion requires knowledge about the K d value of Syd-IMV binding, our IMV re-isolation experiments suggested that the dissociation rate of Syd is considerably high. Thus, real time binding/dissociation measurements will be required to determine the K d value. While the simple steric hindrance discussed above is possible, we believe it is more likely that Syd causes more drastic structural changes to the SecYE complex. Syd was found to inhibit the SecA insertion reactions. We previously noted that the SecY205 mutant form of SecY (32Taura T. Akiyama Y. Ito K. Mol. Gen. Genet. 1994; 243: 261-269Crossref PubMed Scopus (62) Google Scholar) did not allow the preprotein- and ATP-dependent mode of SecA insertion, while it still allowed SecA insertion in the presence of AMP-PNP (5Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar). Our present results show, in contrast, that Syd abolishes both of these modes of SecA insertion reactions. Thus, it is conceivable that the SecY205 defect lies in a specific process of the SecA insertion reaction cascade, whereas Syd impairs the system more generally or inhibits a very early event. Indeed, Syd inhibited the high affinity binding between SecA and the SecY24 IMV. Furthermore, it acted to dissociate the inserted SecA segment that had been stabilized by AMP-PNP. In these cases, the subunit arrangement of the SecYE complex may have been altered, leading to the elimination of both the high affinity SecA binding site and the structural element (or channel) required for maintaining the “inserted” state of SecA. Previous studies suggested that the high affinity binding of SecA requires both SecY and SecE (3Hartl F.-U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (447) Google Scholar, 33Hanada M. Nishiyama K. Mizushima S. Tokuda H. J. Biol. Chem. 1994; 269: 23625-23631Abstract Full Text PDF PubMed Google Scholar, 34Duong F. Wickner W. EMBO J. 1997; 16: 2756-2768Crossref PubMed Scopus (230) Google Scholar). Thus, it seems to be a logical explanation that the presence of a high concentration of Syd acts to dissociate the SecY-SecE complex that is already compromised by the SecY24 alteration. An interesting possibility here is that Syd may bind preferentially to an uncomplexed state of SecY. This is consistent with the known ability of Syd to stabilize overproduced SecY (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Although the physiological role of Syd is elusive (19Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), some speculations may be useful. For instance, Syd may act as a reservoir for unassembled forms of SecY, thus preventing its deleterious effects on the cell (14Kihara A. Akiyama Y. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4532-4536Crossref PubMed Scopus (210) Google Scholar). Alternatively, it may serve as a regulatory factor that negatively controls the translocase function by interfering with either SecY-SecE or SecY-SecA binding. At any rate, interaction between Syd and SecY seems specific in that it is disrupted by the secY249alteration of residue 249 of SecY. Syd will be useful to probe the architecture of protein translocase from the functional point of view. We thank Tohru Yoshihisa and Yoshinori Akiyama for useful comments; Gen Matsumoto for suggestions in the SecA insertion and binding experiments; and Kiyoko Mochizuki, Yusuke Shimizu, and Toshiki Yabe for technical support." @default.
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