Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1990 Sep;9(9):2723–2729. doi: 10.1002/j.1460-2075.1990.tb07459.x

Initial steps in protein membrane insertion. Bacteriophage M13 procoat protein binds to the membrane surface by electrostatic interaction.

A Gallusser 1, A Kuhn 1
PMCID: PMC551979  PMID: 2202592

Abstract

Bacteriophage M13 procoat protein is synthesized on free polysomes prior to its assembly into the inner membrane of Escherichia coli. As an initial step of the membrane insertion pathway, the precursor protein interacts with the cytoplasmic face of the inner membrane. We have used oligonucleotide-directed mutagenesis to study the regions of the procoat protein involved in membrane binding. We find that there is an absolute requirement for positively charged amino acids at both ends of the protein. Replacing these with negatively charged residues resulted in an accumulation of the precursor in the cytoplasm. We propose that the positively charged amino acids are directly involved in membrane binding, possibly directly to the negatively charged phospholipid head groups. This was tested in vitro with artificial liposomes. Whereas wild-type procoat interacted with these liposomes, we found that procoat mutants with negatively charged amino acids at both ends did not bind. Therefore, we conclude that newly synthesized M13 procoat protein binds electrostatically to the negatively charged inner membrane of E. coli.

Full text

PDF
2723

Images in this article

2724Fig. 1.
on p.2724
2725Fig. 2.
on p.2725
2726Fig. 3.
on p.2726
2726Fig. 4.
on p.2726
2727Fig. 5.
on p.2727
2728Fig. 7.
on p.2728

Selected References

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

  1. Akiyama Y., Ito K. The SecY membrane component of the bacterial protein export machinery: analysis by new electrophoretic methods for integral membrane proteins. EMBO J. 1985 Dec 1;4(12):3351–3356. doi: 10.1002/j.1460-2075.1985.tb04088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bedouelle H., Bassford P. J., Jr, Fowler A. V., Zabin I., Beckwith J., Hofnung M. Mutations which alter the function of the signal sequence of the maltose binding protein of Escherichia coli. Nature. 1980 May 8;285(5760):78–81. doi: 10.1038/285078a0. [DOI] [PubMed] [Google Scholar]
  3. Bosch D., de Boer P., Bitter W., Tommassen J. The role of the positively charged N-terminus of the signal sequence of E. coli outer membrane protein PhoE in export. Biochim Biophys Acta. 1989 Feb 13;979(1):69–76. doi: 10.1016/0005-2736(89)90524-5. [DOI] [PubMed] [Google Scholar]
  4. Burnell E., van Alphen L., Verkleij A., de Kruijff B. 31P nuclear magnetic resonance and freeze-fracture electron microscopy studies on Escherichia coli. I. Cytoplasmic membrane and total phospholipids. Biochim Biophys Acta. 1980 Apr 24;597(3):492–501. doi: 10.1016/0005-2736(80)90222-9. [DOI] [PubMed] [Google Scholar]
  5. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Cross T. A., Opella S. J. Protein structure by solid state nuclear magnetic resonance. Residues 40 to 45 of bacteriophage fd coat protein. J Mol Biol. 1985 Apr 5;182(3):367–381. doi: 10.1016/0022-2836(85)90197-4. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Geller B. L., Wickner W. M13 procoat inserts into liposomes in the absence of other membrane proteins. J Biol Chem. 1985 Oct 25;260(24):13281–13285. [PubMed] [Google Scholar]
  11. Iino T., Takahashi M., Sako T. Role of amino-terminal positive charge on signal peptide in staphylokinase export across the cytoplasmic membrane of Escherichia coli. J Biol Chem. 1987 May 25;262(15):7412–7417. [PubMed] [Google Scholar]
  12. Inouye S., Soberon X., Franceschini T., Nakamura K., Itakura K., Inouye M. Role of positive charge on the amino-terminal region of the signal peptide in protein secretion across the membrane. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3438–3441. doi: 10.1073/pnas.79.11.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ito K., Date T., Wickner W. Synthesis, assembly into the cytoplasmic membrane, and proteolytic processing of the precursor of coliphage M13 coat protein. J Biol Chem. 1980 Mar 10;255(5):2123–2130. [PubMed] [Google Scholar]
  14. Koshland D., Botstein D. Evidence for posttranslational translocation of beta-lactamase across the bacterial inner membrane. Cell. 1982 Oct;30(3):893–902. doi: 10.1016/0092-8674(82)90294-x. [DOI] [PubMed] [Google Scholar]
  15. Krieg P. A., Melton D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuhn A. Alterations in the extracellular domain of M13 procoat protein make its membrane insertion dependent on secA and secY. Eur J Biochem. 1988 Nov 1;177(2):267–271. doi: 10.1111/j.1432-1033.1988.tb14372.x. [DOI] [PubMed] [Google Scholar]
  17. Kuhn A. Bacteriophage M13 procoat protein inserts into the plasma membrane as a loop structure. Science. 1987 Dec 4;238(4832):1413–1415. doi: 10.1126/science.3317833. [DOI] [PubMed] [Google Scholar]
  18. Kuhn A., Kreil G., Wickner W. Both hydrophobic domains of M13 procoat are required to initiate membrane insertion. EMBO J. 1986 Dec 20;5(13):3681–3685. doi: 10.1002/j.1460-2075.1986.tb04699.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kuhn A., Wickner W. Conserved residues of the leader peptide are essential for cleavage by leader peptidase. J Biol Chem. 1985 Dec 15;260(29):15914–15918. [PubMed] [Google Scholar]
  20. Kuhn A., Wickner W., Kreil G. The cytoplasmic carboxy terminus of M13 procoat is required for the membrane insertion of its central domain. Nature. 1986 Jul 24;322(6077):335–339. doi: 10.1038/322335a0. [DOI] [PubMed] [Google Scholar]
  21. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lill R., Crooke E., Guthrie B., Wickner W. The "trigger factor cycle" includes ribosomes, presecretory proteins, and the plasma membrane. Cell. 1988 Sep 23;54(7):1013–1018. doi: 10.1016/0092-8674(88)90116-x. [DOI] [PubMed] [Google Scholar]
  23. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ohno-Iwashita Y., Wickner W. Reconstitution of rapid and asymmetric assembly of M13 procoat protein into liposomes which have bacterial leader peptidase. J Biol Chem. 1983 Feb 10;258(3):1895–1900. [PubMed] [Google Scholar]
  25. Oliver D. B., Beckwith J. Regulation of a membrane component required for protein secretion in Escherichia coli. Cell. 1982 Aug;30(1):311–319. doi: 10.1016/0092-8674(82)90037-x. [DOI] [PubMed] [Google Scholar]
  26. Racker E. Reconstitution of membrane processes. Methods Enzymol. 1979;55:699–711. doi: 10.1016/0076-6879(79)55078-2. [DOI] [PubMed] [Google Scholar]
  27. Richardson W. D., Roberts B. L., Smith A. E. Nuclear location signals in polyoma virus large-T. Cell. 1986 Jan 17;44(1):77–85. doi: 10.1016/0092-8674(86)90486-1. [DOI] [PubMed] [Google Scholar]
  28. Russel M., Model P. Filamentous phage pre-coat is an integral membrane protein: analysis by a new method of membrane preparation. Cell. 1982 Jan;28(1):177–184. doi: 10.1016/0092-8674(82)90387-7. [DOI] [PubMed] [Google Scholar]
  29. Schlenstedt G., Zimmermann R. Import of frog prepropeptide GLa into microsomes requires ATP but does not involve docking protein or ribosomes. EMBO J. 1987 Mar;6(3):699–703. doi: 10.1002/j.1460-2075.1987.tb04810.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thom J. R., Randall L. L. Role of the leader peptide of maltose-binding protein in two steps of the export process. J Bacteriol. 1988 Dec;170(12):5654–5661. doi: 10.1128/jb.170.12.5654-5661.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vlasuk G. P., Inouye S., Ito H., Itakura K., Inouye M. Effects of the complete removal of basic amino acid residues from the signal peptide on secretion of lipoprotein in Escherichia coli. J Biol Chem. 1983 Jun 10;258(11):7141–7148. [PubMed] [Google Scholar]
  32. Walter P. Signal recognition. Two receptors act sequentially. 1987 Aug 27-Sep 2Nature. 328(6133):763–764. doi: 10.1038/328763a0. [DOI] [PubMed] [Google Scholar]
  33. Wickner W. T., Lodish H. F. Multiple mechanisms of protein insertion into and across membranes. Science. 1985 Oct 25;230(4724):400–407. doi: 10.1126/science.4048938. [DOI] [PubMed] [Google Scholar]
  34. Wiedmann M., Kurzchalia T. V., Hartmann E., Rapoport T. A. A signal sequence receptor in the endoplasmic reticulum membrane. 1987 Aug 27-Sep 2Nature. 328(6133):830–833. doi: 10.1038/328830a0. [DOI] [PubMed] [Google Scholar]
  35. Wolfe P. B., Rice M., Wickner W. Effects of two sec genes on protein assembly into the plasma membrane of Escherichia coli. J Biol Chem. 1985 Feb 10;260(3):1836–1841. [PubMed] [Google Scholar]
  36. Wolfe P. B., Wickner W., Goodman J. M. Sequence of the leader peptidase gene of Escherichia coli and the orientation of leader peptidase in the bacterial envelope. J Biol Chem. 1983 Oct 10;258(19):12073–12080. [PubMed] [Google Scholar]
  37. Zimmermann R., Mollay C. Import of honeybee prepromelittin into the endoplasmic reticulum. Requirements for membrane insertion, processing, and sequestration. J Biol Chem. 1986 Sep 25;261(27):12889–12895. [PubMed] [Google Scholar]
  38. de Vrije T., de Swart R. L., Dowhan W., Tommassen J., de Kruijff B. Phosphatidylglycerol is involved in protein translocation across Escherichia coli inner membranes. Nature. 1988 Jul 14;334(6178):173–175. doi: 10.1038/334173a0. [DOI] [PubMed] [Google Scholar]
  39. von Heijne G. Net N-C charge imbalance may be important for signal sequence function in bacteria. J Mol Biol. 1986 Nov 20;192(2):287–290. doi: 10.1016/0022-2836(86)90365-7. [DOI] [PubMed] [Google Scholar]

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

RESOURCES