Skip to main content
SpringerLink
Log in

Interfering mutations provide in vivo evidence that Escherichia coli SecE functions in multimeric states

  • Original Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

SecY, SecE and SecG form a heterotrimer, which functions as a protein translocation channel in Escherichia coli. The cytosolic loop of SecE contains a segment that is conserved among different organisms. Here we show that mutational alterations in this segment not only inactivate the SecE function but confer dominant interfering properties on the altered SecE molecule. Such effects were especially evident in mutant cells in which the requirement for SecE function was increased. Overproduction of SecE, but not of SecY, alleviated the dominant negative effects. These results suggest that the inactive SecE molecule sequesters wild-type SecE. It was also found that an amino acid substitution, D112P, in the C-terminal periplasmic region intragenically suppressed the dominant interference. These results are consistent with a notion that there is significant SecE-SecE interaction in vivo, in which the C-terminal region has an important role. The data hence suggest that dimeric SecE participates in the formation of the functional translocation channel.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2A–C.
Fig. 3.
Fig. 4A, B.
Fig. 5.
Fig. 6A, B.
Fig. 7.

Similar content being viewed by others

References

  • Akiyama Y, Ito K (1987) Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli. EMBO J 6:3465–3470

    CAS  PubMed  Google Scholar 

  • Akiyama Y, Ogura T, Ito K (1994) Involvement of FtsH in protein assembly into and through the membrane. I. Mutations that reduce retention efficiency of a cytoplasmic reporter. J Biol Chem 269:5218–5224

    CAS  PubMed  Google Scholar 

  • Baba T, Taura T, Shimoike T, Akiyama Y, Yoshihisa T, Ito K (1994) A cytoplasmic domain is important for the formation of a SecY-SecE translocator complex. Proc Natl Acad Sci USA 91:4539–4543

    CAS  PubMed  Google Scholar 

  • Bessonneau P, Besson V, Collinson I, Duong F (2002) The SecYEG preprotein translocation channel is a conformationally dynamic and dimeric structure. EMBO J 21:995–1003

    CAS  PubMed  Google Scholar 

  • Breyton C, Haase W, Rapoport TA, Kühlbrandt W, Collinson I (2002) Three-dimensional structure of the bacterial protein-translocation complex SecYEG. Nature 418:662–665

    Article  CAS  PubMed  Google Scholar 

  • Chiba S, Akiyama Y, Mori H, Matsuo E, Ito K (2000) Length recognition at the N-terminal tail for the initiation of FtsH-mediated proteolysis. EMBO Rep 1:47–52

    Article  CAS  PubMed  Google Scholar 

  • Collinson I, Breyton C, Duong F, Tziatzios C, Schubert D, Or E, Rapoport T, Kühlbrandt W (2001) Projection structure and oligomeric properties of a bacterial core protein translocase. EMBO J 20:2462–2471

    CAS  PubMed  Google Scholar 

  • Duong F, Wickner W (1999) The PrlA and PrlG phenotypes are caused by a loosened association among the translocase SecYEG subunits. EMBO J 18:3263–3270

    Article  CAS  PubMed  Google Scholar 

  • Flower AM, Osborne RS, Silhavy TJ (1995) The allele-specific synthetic lethality of prlA-prlG double mutants predicts interactive domains of SecY and SecE. EMBO J 14:884–893

    CAS  PubMed  Google Scholar 

  • Homma T, Yoshihisa T, Ito K (1997) Subunit interactions in the Escherichia coli protein translocase: SecE and SecG associate independently with SecY. FEBS Lett 408:11–15

    Article  CAS  PubMed  Google Scholar 

  • Ito K, Akiyama Y (1991) In vivo analysis of integration of membrane proteins in Escherichia coli. Mol Microbiol 5:2243–2253

    CAS  PubMed  Google Scholar 

  • Kaufmann A, Manting EH, Veenendaal AKJ, Driessen AJM, van der Does C (1999) Cysteine-directed cross-linking demonstrates that helix 3 of SecE is close to helix 2 of SecY and helix 3 of a neighboring SecE. Biochemistry 38:9115–9125

    Article  CAS  PubMed  Google Scholar 

  • Kihara A, Akiyama Y, Ito K (1995) FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase subunit. Proc Natl Acad Sci USA 92:4532–4536

    CAS  PubMed  Google Scholar 

  • Manting EH, van der Does C, Remigy H, Engel A, Driessen AJM (2000) SecYEG assembles into a tetramer to form the active protein translocation channel. EMBO J 19:852–861

    CAS  PubMed  Google Scholar 

  • Matsumoto G, Yoshihisa T, 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

    Article  CAS  PubMed  Google Scholar 

  • Matsumoto G, Mori H, Ito K (1998) Roles of SecG in ATP- and SecA-dependent protein translocation. Proc Natl Acad Sci USA 95:13567–13572

    Article  CAS  PubMed  Google Scholar 

  • Matsumoto G, Homma T, Mori H, Ito K (2000) A mutation in secY that causes enhanced SecA insertion and impaired late functions in protein translocation. J Bacteriol 182:3377–3382

    Article  CAS  PubMed  Google Scholar 

  • Matsuo E, Ito K (1998) Genetic analysis of an essential cytoplasmic domain of Escherichia coli SecY based on resistance to Syd, a SecY-interacting protein. Mol Gen Genet 258:240–249

    Article  CAS  PubMed  Google Scholar 

  • Matsuo E, Mori H, Shimoike T, Ito K (1998) Syd, a SecY-interacting protein, excludes SecA from the SecYE complex with an altered SecY24 subunit. J Biol Chem 273:18835–18840

    Article  CAS  PubMed  Google Scholar 

  • Matsuyama S, Akimaru J, Mizushima S (1990) SecE-dependent overproduction of SecY in Escherichia coli. FEBS Lett 269:96–100

    Article  CAS  PubMed  Google Scholar 

  • Meyer TH, Ménétret J-F, Breitling R, Miller KR, Akey CW, Rapoport TA (1999) The bacterial SecY/E translocation complex forms channel-like structures similar to those of the eukaryotic Sec61p complex. J Mol Biol 285:1789–1800

    CAS  PubMed  Google Scholar 

  • Mori H, Ito K (2001) The Sec protein-translocation pathway. Trends Microbiol 9:494–500

    CAS  PubMed  Google Scholar 

  • Murphy CK, Beckwith J (1994) Residues essential for the function of SecE, a membrane component of the Escherichia coli secretion apparatus, are located in a conserved cytoplasmic region. Proc Natl Acad Sci USA 91:2557–2561

    CAS  PubMed  Google Scholar 

  • Nagamori S, Nishiyama K, Tokuda H (2000) Two SecG molecules present in a single protein translocation machinery are functional even after crosslinking. J Biochem 128:129–137

    CAS  PubMed  Google Scholar 

  • Nishiyama K, Mizushima S, Tokuda H (1992) The carboxy-terminal region of SecE interacts with SecY and is functional in the reconstitution of protein translocation activity in Escherichia coli. J Biol Chem 267:7170–7176

    CAS  PubMed  Google Scholar 

  • Nishiyama K, Hanada M, 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

    CAS  PubMed  Google Scholar 

  • Nouwen N, Kruijff B, Tommassen J (1996) prlA suppressors in Escherichia coli relieve the proton electrochemical gradient dependency of translocation of wild-type precursors. Proc Natl Acad Sci USA 93:5953–5957

    Article  CAS  PubMed  Google Scholar 

  • Pohlschröder M, Murphy C, Beckwith J (1996) In vivo analyses of interactions between SecE and SecY, core components of Escherichia coli protein translocation machinery. J Biol Chem 271:19908–19914

    Article  PubMed  Google Scholar 

  • Riggs PD, Derman AI, Beckwith J (1988) A mutation affecting the regulation of a secA-lacZ fusion defines a new sec gene. Genetics 118:571–579

    CAS  PubMed  Google Scholar 

  • Schatz PJ, Bieker KL, Ottemann KM, Silhavy TJ, Beckwith J (1991) One of three transmembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E. coli secretion machinery. EMBO J 10:1749–1757

    CAS  PubMed  Google Scholar 

  • Shimoike T, Akiyama Y, Baba T, Taura T, Ito K (1992) SecY variants that interfere with Escherichia coli protein export in the presence of normal secY. Mol Microbiol 6:1205–1210

    CAS  PubMed  Google Scholar 

  • Shimoike T, Taura T, Kihara A, Yoshihisa T, Akiyama Y, Cannon K, Ito K (1995) Product of a new gene, syd, functionally interacts with SecY when overproduced in Escherichia coli. J Biol Chem 270:5519–5526

    Article  PubMed  Google Scholar 

  • Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

  • Suzuki H, Nishiyama K, Tokuda H (1998) Coupled structure change of SecA and SecG revealed by the synthetic lethality of the secAcsR11and ΔsecG::kan double mutant. Mol Microbiol 29:331–341

    Article  CAS  PubMed  Google Scholar 

  • Taura T, Baba T, Akiyama Y, Ito K (1993) Determinants of the quantity of the stable SecY complex in the Escherichia coli cell. J Bacteriol 175:7771–7775

    CAS  PubMed  Google Scholar 

  • Taura T, Akiyama Y, Ito K (1994) Genetic analysis of SecY: additional export-defective mutations and factors affecting their phenotypes. Mol Gen Genet 243:261–269

    CAS  PubMed  Google Scholar 

  • Taura T, Yoshihisa T, Ito K (1997) Protein translocation functions of Escherichia coli SecY: in vitro characterization of cold-sensitive secY mutants. Biochimie 79:517–521

    Article  CAS  PubMed  Google Scholar 

  • Van der Wolk JPW, Fekkes P, Boorsma A, Huie JL, Silhavy TJ, Driessen AJM (1998) PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA-SecY interaction during the initiation of translocation. EMBO J 17:3631–3639

    Article  PubMed  Google Scholar 

  • Veenendaal A, van der Does C, Driessen A (2001) Mapping the sites of interaction between SecY and SecE by cysteine scanning mutagenesis. J Biol Chem 276:32559–32566

    Article  CAS  PubMed  Google Scholar 

  • Veenendaal A, van der Does C, Driessen AJM (2002) The core of the bacterial translocase harbors a tilted transmembrane segment 3 of SecE. J Biol Chem 277:36640–36645

    Article  PubMed  Google Scholar 

  • Yahr TL, Wickner WT (2000) Evaluating the oligomeric state of SecYEG in preprotein translocase. EMBO J 19:4393–4401

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Yoshinori Akiyama for discussion, and Kiyoko Mochizuki, Toshiki Yabe, Yusuke Shimizu, Mikihiro Yamada, and Michiyo Sano for technical assistance. This work was supported by grants from CREST, JST (Japan Science and Technology Corporation), and from the Ministry of Education, Culture, Sports, Science and Technology, Japan. E. M. was supported by JSPS Fellowships for Young Japanese Scientists

Author information

Author notes

  1. E. Matsuo

    Present address: Department of Chemistry, Graduate School of Science, Nagoya University, 464-8602, Nagoya, Japan

Authors and Affiliations

  1. Institute for Virus Research, Kyoto University, Sakyo-ku, 606-8507, Kyoto, Japan

    E. Matsuo, H. Mori & K. Ito

Authors
  1. E. Matsuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H. Mori
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. Ito
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to K. Ito.

Additional information

Communicated by W. Goebel

Rights and permissions

Reprints and permissions

About this article

Cite this article

Matsuo, E., Mori, H. & Ito, K. Interfering mutations provide in vivo evidence that Escherichia coli SecE functions in multimeric states. Mol Gen Genomics 268, 808–815 (2003). https://doi.org/10.1007/s00438-003-0803-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-003-0803-9

Keywords

Search

Navigation