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. 1979 Apr;76(4):1795–1799. doi: 10.1073/pnas.76.4.1795

Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal membranes.

P Walter, R C Jackson, M M Marcus, V R Lingappa, G Blobel
PMCID: PMC383478  PMID: 109833

Abstract

The ability of microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer into the intravesicular space was investigated by using trypsin as a proteolytic probe. We found that under defined conditions trypsin is able to dissect the translocation activity of microsomal membranes into components that can be separated into two fractions, one soluble and the other membrane bound. The trypsinized membrane fraction has lost its translocation activity. Addition of the trypsin-generated soluble fraction, however, results in reconstitution of translocation activity. These results are compatible with the notion proposed in the signal hypothesis that the translocation activity of the microsomal membrane resides in transmembrane protein(s). We propose that trypsin effects solubilization from the membrane of cytosol-exposed domain(s) involved in recognition of the signal sequence or ribosome or both, leaving behind membrane-integrated domain(s) that provide the environment for the passage of the nascent chain across the membrane. Signal peptidase activity was unaffected by trypsinization of microsomal vesicles consistent with a localization of the active site of this enzyme on the cisternal side of the vesicles.

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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. 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]
  4. Dobberstein B., Blobel G. Functional interaction of plant ribosomes with animal microsomal membranes. Biochem Biophys Res Commun. 1977 Feb 21;74(4):1675–1682. doi: 10.1016/0006-291x(77)90637-4. [DOI] [PubMed] [Google Scholar]
  5. Inouye S., Wang S., Sekizawa J., Halegoua S., Inouye M. Amino acid sequence for the peptide extension on the prolipoprotein of the Escherichia coli outer membrane. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1004–1008. doi: 10.1073/pnas.74.3.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jackson R. C., Blobel G. Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5598–5602. doi: 10.1073/pnas.74.12.5598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kreibich G., Ulrich B. L., Sabatini D. D. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes. J Cell Biol. 1978 May;77(2):464–487. doi: 10.1083/jcb.77.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lingappa V. R., Devillers-Thiery A., Blobel G. Nascent prehormones are intermediates in the biosynthesis of authentic bovine pituitary growth hormone and prolactin. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2432–2436. doi: 10.1073/pnas.74.6.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lingappa V. R., Katz F. N., Lodish H. F., Blobel G. A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function. J Biol Chem. 1978 Dec 25;253(24):8667–8670. [PubMed] [Google Scholar]
  10. Lingappa V. R., Shields D., Woo S. L., Blobel G. Nascent chicken ovalbumin contains the functional equivalent of a signal sequence. J Cell Biol. 1978 Nov;79(2 Pt 1):567–572. doi: 10.1083/jcb.79.2.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mercier J. C., Haze G., Gaye P., Hue D. Amino terminal sequence of the precursor of ovine beta-lactoglobulin. Biochem Biophys Res Commun. 1978 Jun 29;82(4):1236–1245. doi: 10.1016/0006-291x(78)90320-0. [DOI] [PubMed] [Google Scholar]
  12. Palmiter R. D., Gagnon J., Walsh K. A. Ovalbumin: a secreted protein without a transient hydrophobic leader sequence. Proc Natl Acad Sci U S A. 1978 Jan;75(1):94–98. doi: 10.1073/pnas.75.1.94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  14. Scheele G., Dobberstein B., Blobel G. Transfer of proteins across membranes, Biosynthesis in vitro of pretrypsinogen and trypsinogen by cell fractions of canine pancreas. Eur J Biochem. 1978 Jan 16;82(2):593–599. doi: 10.1111/j.1432-1033.1978.tb12055.x. [DOI] [PubMed] [Google Scholar]
  15. Shields D., Blobel G. Cell-free synthesis of fish preproinsulin, and processing by heterologous mammalian microsomal membranes. Proc Natl Acad Sci U S A. 1977 May;74(5):2059–2063. doi: 10.1073/pnas.74.5.2059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Shields D., Blobel G. Efficient cleavage and segregation of nascent presecretory proteins in a reticulocyte lysate supplemented with microsomal membranes. J Biol Chem. 1978 Jun 10;253(11):3753–3756. [PubMed] [Google Scholar]
  17. 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]

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