Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1977 Jul;74(7):2830–2834. doi: 10.1073/pnas.74.7.2830

Extracellular labeling of nascent polypeptides traversing the membrane of Escherichia coli.

W P Smith, P C Tai, R C Thompson, B D Davis
PMCID: PMC431309  PMID: 331317

Abstract

To provide direct evidence for the hypothesis that secreted proteins may traverse membranes as growing chains, we labeled spheroplasts of Escherichia coli with a reagent (acetyl[35S]methionyl methylphosphate sulfone) that reacts with amino groups but does not cross the membrane. After fractionation, about 6% of the label in the membrane-polysome fraction was found to be attached to the polysomes. This attachment was via peptidyl-tRNA, as shown by several tests: release of most of the label from purified polysomes at low Mg2+; subsequent loss of about 25,000 daltons on cleavage by dilute alkali; release by puromycin; and release, accompanied by a marked increase in average molecular weight, on peptide chain completion. Moreover, a significant fraction of the completed chains was identified serologically and by molecular weight as a major periplasmic protein, alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1]. This work provides direct evidence that: (i) secreted proteins thread through the membrane as growing peptide chains; and (ii) membrane-associated polysomes in bacteria are functionally attached to membrane and not merely trapped on disruption of the cell.

Full text

PDF
2830

Images in this article

2833Image
on p.2833

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. Bretscher M. S. Human erythrocyte membranes: specific labelling of surface proteins. J Mol Biol. 1971 Jun 28;58(3):775–781. doi: 10.1016/0022-2836(71)90039-8. [DOI] [PubMed] [Google Scholar]
  4. Cancedda R., Schlesinger M. J. Localization of polyribosomes containing alkaline phosphatase nascent polypeptides on membranes of Escherichia coli. J Bacteriol. 1974 Jan;117(1):290–301. doi: 10.1128/jb.117.1.290-301.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chua N. H., Blobel G., Siekevitz P., Palade G. E. Periodic variations in the ratio of free to thylakoid-bound chloroplast ribosomes during the cell cycle of Chlamydomonas reinhardtii. J Cell Biol. 1976 Nov;71(2):497–514. doi: 10.1083/jcb.71.2.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devillers-Thiery A., Kindt T., Scheele G., Blobel G. Homology in amino-terminal sequence of precursors to pancreatic secretory proteins. Proc Natl Acad Sci U S A. 1975 Dec;72(12):5016–5020. doi: 10.1073/pnas.72.12.5016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Glenn A. R. Production of extracellular proteins by bacteria. Annu Rev Microbiol. 1976;30:41–62. doi: 10.1146/annurev.mi.30.100176.000353. [DOI] [PubMed] [Google Scholar]
  9. Inouye H., Beckwith J. Synthesis and processing of an Escherichia coli alkaline phosphatase precursor in vitro. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1440–1444. doi: 10.1073/pnas.74.4.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. May B. K., Elliott W. H. Characteristics of extracellular protease formation by Bacillus subtilis and its control by amino acid repression. Biochim Biophys Acta. 1968 May 21;157(3):607–615. doi: 10.1016/0005-2787(68)90158-5. [DOI] [PubMed] [Google Scholar]
  12. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  13. Randall L. L., Hardy S. J. Synthesis of exported proteins by membrane-bound polysomes from Escherichia coli. Eur J Biochem. 1977 May 2;75(1):43–53. doi: 10.1111/j.1432-1033.1977.tb11502.x. [DOI] [PubMed] [Google Scholar]
  14. Redman C. M., Sabatini D. D. Vectorial discharge of peptides released by puromycin from attached ribosomes. Proc Natl Acad Sci U S A. 1966 Aug;56(2):608–615. doi: 10.1073/pnas.56.2.608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ron E. Z., Kohler R. E., Davis B. D. Polysomes extracted from Escherichia coli by freeze-thaw-lysozyme lysis. Science. 1966 Sep 2;153(3740):1119–1120. doi: 10.1126/science.153.3740.1119. [DOI] [PubMed] [Google Scholar]
  16. Shapiro A. L., Viñuela E., Maizel J. V., Jr Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 1967 Sep 7;28(5):815–820. doi: 10.1016/0006-291x(67)90391-9. [DOI] [PubMed] [Google Scholar]
  17. Tai P. C., Wallace B. J., Herzog E. L., Davis B. D. Properties of initiation-free polysomes of Escherichia coli. Biochemistry. 1973 Feb;12(4):609–615. doi: 10.1021/bi00728a007. [DOI] [PubMed] [Google Scholar]
  18. Yamamoto S., Lampen J. O. Membrane penicillinase of Bacillus licheniformis 749/C, a phospholipoprotein. J Biol Chem. 1975 Apr 25;250(8):3212–3213. [PubMed] [Google Scholar]
  19. Yamamoto S., Lampen J. O. Membrane penicillinase of Bacillus licheniformis 749/C:sequence and possible repeated tetrapeptide structure of the phospholipopeptide region. Proc Natl Acad Sci U S A. 1976 May;73(5):1457–1461. doi: 10.1073/pnas.73.5.1457. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES