Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Jul 1;17(13):3631–3639. doi: 10.1093/emboj/17.13.3631

PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA-SecY interaction during the initiation of translocation.

J P van der Wolk 1, P Fekkes 1, A Boorsma 1, J L Huie 1, T J Silhavy 1, A J Driessen 1
PMCID: PMC1170699  PMID: 9649433

Abstract

In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase. This multisubunit enzyme consists of a preprotein-binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain. PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence. Inner membranes of the prlA4 strain efficiently translocate Delta8proOmpA, a proOmpA derivative with a non-functional signal sequence. Owing to the signal sequence mutation, Delta8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY. At the ATP-dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase. The prlA4 membranes, however, bind SecA with a much higher affinity than the wild-type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation. It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex.

Full Text

The Full Text of this article is available as a PDF (296.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bieker K. L., Phillips G. J., Silhavy T. J. The sec and prl genes of Escherichia coli. J Bioenerg Biomembr. 1990 Jun;22(3):291–310. doi: 10.1007/BF00763169. [DOI] [PubMed] [Google Scholar]
  2. Bost S., Belin D. prl mutations in the Escherichia coli secG gene. J Biol Chem. 1997 Feb 14;272(7):4087–4093. doi: 10.1074/jbc.272.7.4087. [DOI] [PubMed] [Google Scholar]
  3. Brundage L., Hendrick J. P., Schiebel E., Driessen A. J., Wickner W. The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell. 1990 Aug 24;62(4):649–657. doi: 10.1016/0092-8674(90)90111-q. [DOI] [PubMed] [Google Scholar]
  4. Cabelli R. J., Chen L., Tai P. C., Oliver D. B. SecA protein is required for secretory protein translocation into E. coli membrane vesicles. Cell. 1988 Nov 18;55(4):683–692. doi: 10.1016/0092-8674(88)90227-9. [DOI] [PubMed] [Google Scholar]
  5. Chang C. N., Blobel G., Model P. Detection of prokaryotic signal peptidase in an Escherichia coli membrane fraction: endoproteolytic cleavage of nascent f1 pre-coat protein. Proc Natl Acad Sci U S A. 1978 Jan;75(1):361–365. doi: 10.1073/pnas.75.1.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crooke E., Guthrie B., Lecker S., Lill R., Wickner W. ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle. Cell. 1988 Sep 23;54(7):1003–1011. doi: 10.1016/0092-8674(88)90115-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crooke E., Wickner W. Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5216–5220. doi: 10.1073/pnas.84.15.5216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cunningham K., Lill R., Crooke E., Rice M., Moore K., Wickner W., Oliver D. SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA. EMBO J. 1989 Mar;8(3):955–959. doi: 10.1002/j.1460-2075.1989.tb03457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cunningham K., Wickner W. Specific recognition of the leader region of precursor proteins is required for the activation of translocation ATPase of Escherichia coli. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8630–8634. doi: 10.1073/pnas.86.22.8630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Vrije T., Tommassen J., De Kruijff B. Optimal posttranslational translocation of the precursor of PhoE protein across Escherichia coli membrane vesicles requires both ATP and the protonmotive force. Biochim Biophys Acta. 1987 Jun 12;900(1):63–72. doi: 10.1016/0005-2736(87)90278-1. [DOI] [PubMed] [Google Scholar]
  11. Deng W. P., Nickoloff J. A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. doi: 10.1016/0003-2697(92)90280-k. [DOI] [PubMed] [Google Scholar]
  12. Derman A. I., Puziss J. W., Bassford P. J., Jr, Beckwith J. A signal sequence is not required for protein export in prlA mutants of Escherichia coli. EMBO J. 1993 Mar;12(3):879–888. doi: 10.1002/j.1460-2075.1993.tb05728.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Driessen A. J., Fekkes P., van der Wolk J. P. The Sec system. Curr Opin Microbiol. 1998 Apr;1(2):216–222. doi: 10.1016/s1369-5274(98)80014-3. [DOI] [PubMed] [Google Scholar]
  14. Driessen A. J. How proteins cross the bacterial cytoplasmic membrane. J Membr Biol. 1994 Nov;142(2):145–159. doi: 10.1007/BF00234937. [DOI] [PubMed] [Google Scholar]
  15. Driessen A. J. Precursor protein translocation by the Escherichia coli translocase is directed by the protonmotive force. EMBO J. 1992 Mar;11(3):847–853. doi: 10.1002/j.1460-2075.1992.tb05122.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Duong F., Wickner W. Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme. EMBO J. 1997 May 15;16(10):2756–2768. doi: 10.1093/emboj/16.10.2756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Duong F., Wickner W. The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling. EMBO J. 1997 Aug 15;16(16):4871–4879. doi: 10.1093/emboj/16.16.4871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Economou A., Pogliano J. A., Beckwith J., Oliver D. B., Wickner W. SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF. Cell. 1995 Dec 29;83(7):1171–1181. doi: 10.1016/0092-8674(95)90143-4. [DOI] [PubMed] [Google Scholar]
  19. Economou A., Wickner W. SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell. 1994 Sep 9;78(5):835–843. doi: 10.1016/s0092-8674(94)90582-7. [DOI] [PubMed] [Google Scholar]
  20. Emr S. D., Hanley-Way S., Silhavy T. J. Suppressor mutations that restore export of a protein with a defective signal sequence. Cell. 1981 Jan;23(1):79–88. doi: 10.1016/0092-8674(81)90272-5. [DOI] [PubMed] [Google Scholar]
  21. Fekkes P., den Blaauwen T., Driessen A. J. Diffusion-limited interaction between unfolded polypeptides and the Escherichia coli chaperone SecB. Biochemistry. 1995 Aug 8;34(31):10078–10085. doi: 10.1021/bi00031a032. [DOI] [PubMed] [Google Scholar]
  22. Fekkes P., van der Does C., Driessen A. J. The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of precursor protein translocation. EMBO J. 1997 Oct 15;16(20):6105–6113. doi: 10.1093/emboj/16.20.6105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fikes J. D., Bassford P. J., Jr Novel secA alleles improve export of maltose-binding protein synthesized with a defective signal peptide. J Bacteriol. 1989 Jan;171(1):402–409. doi: 10.1128/jb.171.1.402-409.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Flower A. M., Doebele R. C., Silhavy T. J. PrlA and PrlG suppressors reduce the requirement for signal sequence recognition. J Bacteriol. 1994 Sep;176(18):5607–5614. doi: 10.1128/jb.176.18.5607-5614.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Guzman L. M., Belin D., Carson M. J., Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121–4130. doi: 10.1128/jb.177.14.4121-4130.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hanada M., Nishiyama K. I., Mizushima S., Tokuda H. Reconstitution of an efficient protein translocation machinery comprising SecA and the three membrane proteins, SecY, SecE, and SecG (p12). J Biol Chem. 1994 Sep 23;269(38):23625–23631. [PubMed] [Google Scholar]
  27. Hartl F. U., Lecker S., Schiebel E., Hendrick J. P., Wickner W. The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane. Cell. 1990 Oct 19;63(2):269–279. doi: 10.1016/0092-8674(90)90160-g. [DOI] [PubMed] [Google Scholar]
  28. Huie J. L., Silhavy T. J. Suppression of signal sequence defects and azide resistance in Escherichia coli commonly result from the same mutations in secA. J Bacteriol. 1995 Jun;177(12):3518–3526. doi: 10.1128/jb.177.12.3518-3526.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Joly J. C., Wickner W. The SecA and SecY subunits of translocase are the nearest neighbors of a translocating preprotein, shielding it from phospholipids. EMBO J. 1993 Jan;12(1):255–263. doi: 10.1002/j.1460-2075.1993.tb05651.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kimura E., Akita M., Matsuyama S., Mizushima S. Determination of a region in SecA that interacts with presecretory proteins in Escherichia coli. J Biol Chem. 1991 Apr 5;266(10):6600–6606. [PubMed] [Google Scholar]
  31. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  32. Lecker S. H., Driessen A. J., Wickner W. ProOmpA contains secondary and tertiary structure prior to translocation and is shielded from aggregation by association with SecB protein. EMBO J. 1990 Jul;9(7):2309–2314. doi: 10.1002/j.1460-2075.1990.tb07402.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lill R., Cunningham K., Brundage L. A., Ito K., Oliver D., Wickner W. SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli. EMBO J. 1989 Mar;8(3):961–966. doi: 10.1002/j.1460-2075.1989.tb03458.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lill R., Dowhan W., Wickner W. The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell. 1990 Jan 26;60(2):271–280. doi: 10.1016/0092-8674(90)90742-w. [DOI] [PubMed] [Google Scholar]
  35. Manting E. H., van der Does C., Driessen A. J. In vivo cross-linking of the SecA and SecY subunits of the Escherichia coli preprotein translocase. J Bacteriol. 1997 Sep;179(18):5699–5704. doi: 10.1128/jb.179.18.5699-5704.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Matsumoto G., Yoshihisa T., Ito K. SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane. EMBO J. 1997 Nov 3;16(21):6384–6393. doi: 10.1093/emboj/16.21.6384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mitchell C., Oliver D. Two distinct ATP-binding domains are needed to promote protein export by Escherichia coli SecA ATPase. Mol Microbiol. 1993 Nov;10(3):483–497. doi: 10.1111/j.1365-2958.1993.tb00921.x. [DOI] [PubMed] [Google Scholar]
  38. Nouwen N., de Kruijff B., Tommassen J. prlA suppressors in Escherichia coli relieve the proton electrochemical gradient dependency of translocation of wild-type precursors. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5953–5957. doi: 10.1073/pnas.93.12.5953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Oliver D. B., Cabelli R. J., Dolan K. M., Jarosik G. P. Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8227–8231. doi: 10.1073/pnas.87.21.8227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Osborne R. S., Silhavy T. J. PrlA suppressor mutations cluster in regions corresponding to three distinct topological domains. EMBO J. 1993 Sep;12(9):3391–3398. doi: 10.1002/j.1460-2075.1993.tb06013.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pogliano J. A., Beckwith J. SecD and SecF facilitate protein export in Escherichia coli. EMBO J. 1994 Feb 1;13(3):554–561. doi: 10.1002/j.1460-2075.1994.tb06293.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Prinz W. A., Spiess C., Ehrmann M., Schierle C., Beckwith J. Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase. EMBO J. 1996 Oct 1;15(19):5209–5217. [PMC free article] [PubMed] [Google Scholar]
  43. Sako T., Iino T. Distinct mutation sites in prlA suppressor mutant strains of Escherichia coli respond either to suppression of signal peptide mutations or to blockage of staphylokinase processing. J Bacteriol. 1988 Nov;170(11):5389–5391. doi: 10.1128/jb.170.11.5389-5391.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schiebel E., Driessen A. J., Hartl F. U., Wickner W. Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase. Cell. 1991 Mar 8;64(5):927–939. doi: 10.1016/0092-8674(91)90317-r. [DOI] [PubMed] [Google Scholar]
  45. Shiozuka K., Tani K., Mizushima S., Tokuda H. The proton motive force lowers the level of ATP required for the in vitro translocation of a secretory protein in Escherichia coli. J Biol Chem. 1990 Nov 5;265(31):18843–18847. [PubMed] [Google Scholar]
  46. Snyders S., Ramamurthy V., Oliver D. Identification of a region of interaction between Escherichia coli SecA and SecY proteins. J Biol Chem. 1997 Apr 25;272(17):11302–11306. doi: 10.1074/jbc.272.17.11302. [DOI] [PubMed] [Google Scholar]
  47. Stader J., Gansheroff L. J., Silhavy T. J. New suppressors of signal-sequence mutations, prlG, are linked tightly to the secE gene of Escherichia coli. Genes Dev. 1989 Jul;3(7):1045–1052. doi: 10.1101/gad.3.7.1045. [DOI] [PubMed] [Google Scholar]
  48. Tanji Y., Gennity J., Pollitt S., Inouye M. Effect of OmpA signal peptide mutations on OmpA secretion, synthesis, and assembly. J Bacteriol. 1991 Mar;173(6):1997–2005. doi: 10.1128/jb.173.6.1997-2005.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Uchida K., Mori H., Mizushima S. Stepwise movement of preproteins in the process of translocation across the cytoplasmic membrane of Escherichia coli. J Biol Chem. 1995 Dec 29;270(52):30862–30868. doi: 10.1074/jbc.270.52.30862. [DOI] [PubMed] [Google Scholar]
  50. Weiss J. B., Ray P. H., Bassford P. J., Jr Purified secB protein of Escherichia coli retards folding and promotes membrane translocation of the maltose-binding protein in vitro. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8978–8982. doi: 10.1073/pnas.85.23.8978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wickner W., Driessen A. J., Hartl F. U. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochem. 1991;60:101–124. doi: 10.1146/annurev.bi.60.070191.000533. [DOI] [PubMed] [Google Scholar]
  52. Yamada H., Matsuyama S., Tokuda H., Mizushima S. A high concentration of SecA allows proton motive force-independent translocation of a model secretory protein into Escherichia coli membrane vesicles. J Biol Chem. 1989 Nov 5;264(31):18577–18581. [PubMed] [Google Scholar]
  53. den Blaauwen T., Fekkes P., de Wit J. G., Kuiper W., Driessen A. J. Domain interactions of the peripheral preprotein Translocase subunit SecA. Biochemistry. 1996 Sep 17;35(37):11994–12004. doi: 10.1021/bi9605088. [DOI] [PubMed] [Google Scholar]
  54. den Blaauwen T., de Wit J. G., Gosker H., van der Does C., Breukink E. J., de Leij L., Driessen A. J. Inhibition of preprotein translocation and reversion of the membrane inserted state of SecA by a carboxyl terminus binding mAb. Biochemistry. 1997 Jul 29;36(30):9159–9168. doi: 10.1021/bi970344a. [DOI] [PubMed] [Google Scholar]
  55. van der Does C., Manting E. H., Kaufmann A., Lutz M., Driessen A. J. Interaction between SecA and SecYEG in micellar solution and formation of the membrane-inserted state. Biochemistry. 1998 Jan 6;37(1):201–210. doi: 10.1021/bi972105t. [DOI] [PubMed] [Google Scholar]
  56. van der Wolk J. P., de Wit J. G., Driessen A. J. The catalytic cycle of the escherichia coli SecA ATPase comprises two distinct preprotein translocation events. EMBO J. 1997 Dec 15;16(24):7297–7304. doi: 10.1093/emboj/16.24.7297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. van der Wolk J., Klose M., Breukink E., Demel R. A., de Kruijff B., Freudl R., Driessen A. J. Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane. Mol Microbiol. 1993 Apr;8(1):31–42. doi: 10.1111/j.1365-2958.1993.tb01200.x. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES