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. 1994 Sep;176(18):5607–5614. doi: 10.1128/jb.176.18.5607-5614.1994

PrlA and PrlG suppressors reduce the requirement for signal sequence recognition.

A M Flower 1, R C Doebele 1, T J Silhavy 1
PMCID: PMC196762  PMID: 8083155

Abstract

Selection for suppressors of defects in the signal sequence of secretory proteins has led most commonly to identification of prlA alleles and less often to identification of prlG alleles. These genes, secY/prlA and secE/prlG, encode integral membrane components of the protein translocation system of Escherichia coli. We demonstrate that an outer membrane protein, LamB, that lacks a signal sequence can be exported with reasonable efficiency in both prlA and prlG suppressor strains. Although the signal sequence is not absolutely required for export of LamB, the level of export in the absence of prl suppressor alleles is exceedingly low. Such strains are phenotypically LamB-, and functional LamB can be detected only by using sensitive infectious-center assays. Suppression of the LamB signal sequence deletion is dependent on normal components of the export pathway, indicating that suppression is not occurring through a bypass mechanism. Our results indicate that the majority of the known prlA suppressors function by an identical mechanism and, further, that the prlG suppressors work in a similar fashion. We propose that both PrlA and PrlG suppressors lack a proofreading activity that normally rejects defective precursors from the export pathway.

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Selected References

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  1. Akimaru J., Matsuyama S., Tokuda H., Mizushima S. Reconstitution of a protein translocation system containing purified SecY, SecE, and SecA from Escherichia coli. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6545–6549. doi: 10.1073/pnas.88.15.6545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akita M., Sasaki S., Matsuyama S., Mizushima S. SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli. J Biol Chem. 1990 May 15;265(14):8164–8169. [PubMed] [Google Scholar]
  3. Akiyama Y., Ito K. Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli. EMBO J. 1987 Nov;6(11):3465–3470. doi: 10.1002/j.1460-2075.1987.tb02670.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Altman E., Bankaitis V. A., Emr S. D. Characterization of a region in mature LamB protein that interacts with a component of the export machinery of Escherichia coli. J Biol Chem. 1990 Oct 25;265(30):18148–18153. [PubMed] [Google Scholar]
  5. Altman E., Emr S. D., Kumamoto C. A. The presence of both the signal sequence and a region of mature LamB protein is required for the interaction of LamB with the export factor SecB. J Biol Chem. 1990 Oct 25;265(30):18154–18160. [PubMed] [Google Scholar]
  6. Arkowitz R. A., Wickner W. SecD and SecF are required for the proton electrochemical gradient stimulation of preprotein translocation. EMBO J. 1994 Feb 15;13(4):954–963. doi: 10.1002/j.1460-2075.1994.tb06340.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bankaitis V. A., Bassford P. J., Jr Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope. J Bacteriol. 1985 Jan;161(1):169–178. doi: 10.1128/jb.161.1.169-178.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bieker-Brady K., Silhavy T. J. Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli. EMBO J. 1992 Sep;11(9):3165–3174. doi: 10.1002/j.1460-2075.1992.tb05393.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Bieker K. L., Silhavy T. J. PrlA (SecY) and PrlG (SecE) interact directly and function sequentially during protein translocation in E. coli. Cell. 1990 Jun 1;61(5):833–842. doi: 10.1016/0092-8674(90)90193-i. [DOI] [PubMed] [Google Scholar]
  11. Bowden G. A., Baneyx F., Georgiou G. Abnormal fractionation of beta-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide. J Bacteriol. 1992 May;174(10):3407–3410. doi: 10.1128/jb.174.10.3407-3410.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Brundage L., Fimmel C. J., Mizushima S., Wickner W. SecY, SecE, and band 1 form the membrane-embedded domain of Escherichia coli preprotein translocase. J Biol Chem. 1992 Feb 25;267(6):4166–4170. [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Carlson J. H., Silhavy T. J. Signal sequence processing is required for the assembly of LamB trimers in the outer membrane of Escherichia coli. J Bacteriol. 1993 Jun;175(11):3327–3334. doi: 10.1128/jb.175.11.3327-3334.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  17. Cerretti D. P., Dean D., Davis G. R., Bedwell D. M., Nomura M. The spc ribosomal protein operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene. Nucleic Acids Res. 1983 May 11;11(9):2599–2616. doi: 10.1093/nar/11.9.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  19. Collier D. N., Bankaitis V. A., Weiss J. B., Bassford P. J., Jr The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein. Cell. 1988 Apr 22;53(2):273–283. doi: 10.1016/0092-8674(88)90389-3. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Douville K., Leonard M., Brundage L., Nishiyama K., Tokuda H., Mizushima S., Wickner W. Band 1 subunit of Escherichia coli preportein translocase and integral membrane export factor P12 are the same protein. J Biol Chem. 1994 Jul 22;269(29):18705–18707. [PubMed] [Google Scholar]
  22. Downing W. L., Sullivan S. L., Gottesman M. E., Dennis P. P. Sequence and transcriptional pattern of the essential Escherichia coli secE-nusG operon. J Bacteriol. 1990 Mar;172(3):1621–1627. doi: 10.1128/jb.172.3.1621-1627.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Emr S. D., Hedgpeth J., Clément J. M., Silhavy T. J., Hofnung M. Sequence analysis of mutations that prevent export of lambda receptor, an Escherichia coli outer membrane protein. Nature. 1980 May 8;285(5760):82–85. doi: 10.1038/285082a0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Gannon P. M., Li P., Kumamoto C. A. The mature portion of Escherichia coli maltose-binding protein (MBP) determines the dependence of MBP on SecB for export. J Bacteriol. 1989 Feb;171(2):813–818. doi: 10.1128/jb.171.2.813-818.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Gold L., Pribnow D., Schneider T., Shinedling S., Singer B. S., Stormo G. Translational initiation in prokaryotes. Annu Rev Microbiol. 1981;35:365–403. doi: 10.1146/annurev.mi.35.100181.002053. [DOI] [PubMed] [Google Scholar]
  28. Hardy S. J., Randall L. L. A kinetic partitioning model of selective binding of nonnative proteins by the bacterial chaperone SecB. Science. 1991 Jan 25;251(4992):439–443. doi: 10.1126/science.1989077. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Jones J. D., McKnight C. J., Gierasch L. M. Biophysical studies of signal peptides: implications for signal sequence functions and the involvement of lipid in protein export. J Bioenerg Biomembr. 1990 Jun;22(3):213–232. doi: 10.1007/BF00763166. [DOI] [PubMed] [Google Scholar]
  31. Kumamoto C. A. Escherichia coli SecB protein associates with exported protein precursors in vivo. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5320–5324. doi: 10.1073/pnas.86.14.5320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kumamoto C. A., Gannon P. M. Effects of Escherichia coli secB mutations on pre-maltose binding protein conformation and export kinetics. J Biol Chem. 1988 Aug 15;263(23):11554–11558. [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. Liu G., Topping T. B., Randall L. L. Physiological role during export for the retardation of folding by the leader peptide of maltose-binding protein. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9213–9217. doi: 10.1073/pnas.86.23.9213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Matsuyama S., Fujita Y., Mizushima S. SecD is involved in the release of translocated secretory proteins from the cytoplasmic membrane of Escherichia coli. EMBO J. 1993 Jan;12(1):265–270. doi: 10.1002/j.1460-2075.1993.tb05652.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Misra R., Peterson A., Ferenci T., Silhavy T. J. A genetic approach for analyzing the pathway of LamB assembly into the outer membrane of Escherichia coli. J Biol Chem. 1991 Jul 25;266(21):13592–13597. [PubMed] [Google Scholar]
  37. Nishiyama K., Kabuyama Y., Akimaru J., Matsuyama S., Tokuda H., Mizushima S. SecY is an indispensable component of the protein secretory machinery of Escherichia coli. Biochim Biophys Acta. 1991 May 31;1065(1):89–97. doi: 10.1016/0005-2736(91)90015-z. [DOI] [PubMed] [Google Scholar]
  38. Nishiyama K., Mizushima S., Tokuda H. A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli. EMBO J. 1993 Sep;12(9):3409–3415. doi: 10.1002/j.1460-2075.1993.tb06015.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Park S., Liu G., Topping T. B., Cover W. H., Randall L. L. Modulation of folding pathways of exported proteins by the leader sequence. Science. 1988 Feb 26;239(4843):1033–1035. doi: 10.1126/science.3278378. [DOI] [PubMed] [Google Scholar]
  41. Puziss J. W., Fikes J. D., Bassford P. J., Jr Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide. J Bacteriol. 1989 May;171(5):2303–2311. doi: 10.1128/jb.171.5.2303-2311.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Randall L. L. Peptide binding by chaperone SecB: implications for recognition of nonnative structure. Science. 1992 Jul 10;257(5067):241–245. doi: 10.1126/science.1631545. [DOI] [PubMed] [Google Scholar]
  43. Randall L. L., Topping T. B., Hardy S. J. No specific recognition of leader peptide by SecB, a chaperone involved in protein export. Science. 1990 May 18;248(4957):860–863. doi: 10.1126/science.2188362. [DOI] [PubMed] [Google Scholar]
  44. Russo F. D., Slauch J. M., Silhavy T. J. Mutations that affect separate functions of OmpR the phosphorylated regulator of porin transcription in Escherichia coli. J Mol Biol. 1993 May 20;231(2):261–273. doi: 10.1006/jmbi.1993.1281. [DOI] [PubMed] [Google Scholar]
  45. Schatz P. J., Beckwith J. Genetic analysis of protein export in Escherichia coli. Annu Rev Genet. 1990;24:215–248. doi: 10.1146/annurev.ge.24.120190.001243. [DOI] [PubMed] [Google Scholar]
  46. Schatz P. J., Bieker K. L., Ottemann K. M., Silhavy T. J., Beckwith J. 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. 1991 Jul;10(7):1749–1757. doi: 10.1002/j.1460-2075.1991.tb07699.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schatz P. J., Riggs P. D., Jacq A., Fath M. J., Beckwith J. The secE gene encodes an integral membrane protein required for protein export in Escherichia coli. Genes Dev. 1989 Jul;3(7):1035–1044. doi: 10.1101/gad.3.7.1035. [DOI] [PubMed] [Google Scholar]
  48. Snyder W. B., Silhavy T. J. Enhanced export of beta-galactosidase fusion proteins in prlF mutants is Lon dependent. J Bacteriol. 1992 Sep;174(17):5661–5668. doi: 10.1128/jb.174.17.5661-5668.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stader J., Benson S. A., Silhavy T. J. Kinetic analysis of lamB mutants suggests the signal sequence plays multiple roles in protein export. J Biol Chem. 1986 Nov 15;261(32):15075–15080. [PubMed] [Google Scholar]
  50. 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]
  51. Trun N. J., Silhavy T. J. Characterization and in vivo cloning of prlC, a suppressor of signal sequence mutations in Escherichia coli K12. Genetics. 1987 Aug;116(4):513–521. doi: 10.1093/genetics/116.4.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Watanabe M., Blobel G. SecA protein is required for translocation of a model precursor protein into inverted vesicles of Escherichia coli plasma membrane. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9011–9015. doi: 10.1073/pnas.90.19.9011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Watanabe M., Blobel G. SecB functions as a cytosolic signal recognition factor for protein export in E. coli. Cell. 1989 Aug 25;58(4):695–705. doi: 10.1016/0092-8674(89)90104-9. [DOI] [PubMed] [Google Scholar]
  54. Wei S. Q., Stader J. Two distinct regions of the LamB signal sequence function in different steps in export. J Biol Chem. 1994 Jan 21;269(3):1648–1653. [PubMed] [Google Scholar]
  55. Weiss J. B., Bassford P. J., Jr The folding properties of the Escherichia coli maltose-binding protein influence its interaction with SecB in vitro. J Bacteriol. 1990 Jun;172(6):3023–3029. doi: 10.1128/jb.172.6.3023-3029.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. 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]
  57. de Cock H., Overeem W., Tommassen J. Biogenesis of outer membrane protein PhoE of Escherichia coli. Evidence for multiple SecB-binding sites in the mature portion of the PhoE protein. J Mol Biol. 1992 Mar 20;224(2):369–379. doi: 10.1016/0022-2836(92)91001-6. [DOI] [PubMed] [Google Scholar]

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