Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1982;1(5):573–578. doi: 10.1002/j.1460-2075.1982.tb01210.x

The purification of M13 procoat, a membrane protein precursor.

C Zwizinski, W Wickner
PMCID: PMC553089  PMID: 6765231

Abstract

Many membrane proteins and most secreted proteins are initially made as precursors with an N-terminal leader sequence. We now report the isolation of M13 procoat, the precursor of the membrane-bound form of M13 coat protein. There are 40 000 copies of M13 procoat protein/cell during M13 amber 7 virus infection. Purified procoat is quantitatively cleaved by isolated leader peptidase to yield mature-length coat protein. Rabbit antibodies to M13 procoat will precipitate procoat but not coat, suggesting that the antibody molecules are specifically recognizing the leader sequence or the conformation which it induces in the whole procoat molecule.

Full text

PDF
573

Images in this article

575Fig. 2.
on p.575
576Fig. 4.
on p.576

Selected References

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

  1. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ito K., Mandel G., Wickner W. Soluble precursor of an integral membrane protein: synthesis of procoat protein in Escherichia coli infected with bacteriophage M13. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1199–1203. doi: 10.1073/pnas.76.3.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Milstein C., Brownlee G. G., Harrison T. M., Mathews M. B. A possible precursor of immunoglobulin light chains. Nat New Biol. 1972 Sep 27;239(91):117–120. doi: 10.1038/newbio239117a0. [DOI] [PubMed] [Google Scholar]
  5. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  6. Ohkawa I., Webster R. E. The orientation of the major coat protein of bacteriophage f1 in the cytoplasmic membrane of Escherichia coli. J Biol Chem. 1981 Oct 10;256(19):9951–9958. [PubMed] [Google Scholar]
  7. Rose J. K., Shafferman A. Conditional expression of the vesicular stomatitis virus glycoprotein gene in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6670–6674. doi: 10.1073/pnas.78.11.6670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Silver P., Watts C., Wickner W. Membrane assembly from purified components. I. Isolated M13 procoat does not require ribosomes or soluble proteins for processing by membranes. Cell. 1981 Aug;25(2):341–345. doi: 10.1016/0092-8674(81)90052-0. [DOI] [PubMed] [Google Scholar]
  10. Walter P., Jackson R. C., Marcus M. M., Lingappa V. R., Blobel G. Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal membranes. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1795–1799. doi: 10.1073/pnas.76.4.1795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Warren G., Dobberstein B. Protein transfer across microsomal membranes reassembled from separated membrane components. Nature. 1978 Jun 15;273(5663):569–571. doi: 10.1038/273569a0. [DOI] [PubMed] [Google Scholar]
  12. Watts C., Silver P., Wickner W. Membrane assembly from purified components. II. Assembly of M13 procoat into liposomes reconstituted with purified leader peptidase. Cell. 1981 Aug;25(2):347–353. doi: 10.1016/0092-8674(81)90053-2. [DOI] [PubMed] [Google Scholar]
  13. Wickner W. Assembly of proteins into membranes. Science. 1980 Nov 21;210(4472):861–868. doi: 10.1126/science.7001628. [DOI] [PubMed] [Google Scholar]
  14. Wickner W. Asymmetric orientation of a phage coat protein in cytoplasmic membrane of Escherichia coli. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4749–4753. doi: 10.1073/pnas.72.12.4749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Wickner W. Asymmetric orientation of phage M13 coat protein in Escherichia coli cytoplasmic membranes and in synthetic lipid vesicles. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1159–1163. doi: 10.1073/pnas.73.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wickner W., Killick T. Membrane-associated assembly of M13 phage in extracts of virus-infected Escherichia coli. Proc Natl Acad Sci U S A. 1977 Feb;74(2):505–509. doi: 10.1073/pnas.74.2.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Zwizinski C., Wickner W. Purification and characterization of leader (signal) peptidase from Escherichia coli. J Biol Chem. 1980 Aug 25;255(16):7973–7977. [PubMed] [Google Scholar]

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

RESOURCES