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. 1988 Jun;7(6):1769–1775. doi: 10.1002/j.1460-2075.1988.tb03007.x

The affinity of signal recognition particle for presecretory proteins is dependent on nascent chain length.

V Siegel 1, P Walter 1
PMCID: PMC457167  PMID: 3169004

Abstract

We have developed an assay in which incomplete preprolactin chains of varying lengths are targeted to the endoplasmic reticulum (ER) membrane in an elongation independent manner. The reaction had the same molecular requirements as nascent chain translocation across the ER membrane, namely, it was signal recognition particle (SRP) dependent, and required the nascent chain to be present as peptidyl tRNA (i.e. most likely ribosome associated) and to have its signal sequence exposed outside the ribosome. We found that the efficiency of the targeting reaction dropped dramatically as the chains grew longer than 140 amino acids in length, which probably reflected a decrease in affinity of the nascent chain-ribosome complex for SRP. Thus at physiological SRP concentrations (10 nM) there appears a sharp cut-off point in the ability of these chains to be targeted, while at high SRP concentrations (270 nM) all chains could be targeted. In kinetic experiments, high concentrations of SRP were found to change the time in elongation after which translocation of the nascent polypeptide could no longer occur.

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Selected References

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

  1. Ainger K. J., Meyer D. I. Translocation of nascent secretory proteins across membranes can occur late in translation. EMBO J. 1986 May;5(5):951–955. doi: 10.1002/j.1460-2075.1986.tb04308.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Caulfield M. P., Duong L. T., Rosenblatt M. Demonstration of post-translational secretion of human placental lactogen by a mammalian in vitro translation system. J Biol Chem. 1986 Aug 25;261(24):10953–10956. [PubMed] [Google Scholar]
  3. Connolly T., Gilmore R. Formation of a functional ribosome-membrane junction during translocation requires the participation of a GTP-binding protein. J Cell Biol. 1986 Dec;103(6 Pt 1):2253–2261. doi: 10.1083/jcb.103.6.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Erickson A. H., Blobel G. Cell-free translation of messenger RNA in a wheat germ system. Methods Enzymol. 1983;96:38–50. doi: 10.1016/s0076-6879(83)96007-x. [DOI] [PubMed] [Google Scholar]
  5. Gilmore R., Blobel G. Transient involvement of signal recognition particle and its receptor in the microsomal membrane prior to protein translocation. Cell. 1983 Dec;35(3 Pt 2):677–685. doi: 10.1016/0092-8674(83)90100-9. [DOI] [PubMed] [Google Scholar]
  6. Gilmore R., Blobel G. Translocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants. Cell. 1985 Sep;42(2):497–505. doi: 10.1016/0092-8674(85)90107-2. [DOI] [PubMed] [Google Scholar]
  7. Gilmore R., Walter P., Blobel G. Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor. J Cell Biol. 1982 Nov;95(2 Pt 1):470–477. doi: 10.1083/jcb.95.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hansen W., Garcia P. D., Walter P. In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor. Cell. 1986 May 9;45(3):397–406. doi: 10.1016/0092-8674(86)90325-9. [DOI] [PubMed] [Google Scholar]
  9. Hobden A. N., Cundliffe E. The mode of action of alpha sarcin and a novel assay of the puromycin reaction. Biochem J. 1978 Jan 15;170(1):57–61. doi: 10.1042/bj1700057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Meyer D. I., Krause E., Dobberstein B. Secretory protein translocation across membranes-the role of the "docking protein'. Nature. 1982 Jun 24;297(5868):647–650. doi: 10.1038/297647a0. [DOI] [PubMed] [Google Scholar]
  11. Mueckler M., Lodish H. F. Post-translational insertion of a fragment of the glucose transporter into microsomes requires phosphoanhydride bond cleavage. Nature. 1986 Aug 7;322(6079):549–552. doi: 10.1038/322549a0. [DOI] [PubMed] [Google Scholar]
  12. Perara E., Rothman R. E., Lingappa V. R. Uncoupling translocation from translation: implications for transport of proteins across membranes. Science. 1986 Apr 18;232(4748):348–352. doi: 10.1126/science.3961485. [DOI] [PubMed] [Google Scholar]
  13. Rothblatt J. A., Meyer D. I. Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-factor and invertase in a homologous cell-free system. Cell. 1986 Feb 28;44(4):619–628. doi: 10.1016/0092-8674(86)90271-0. [DOI] [PubMed] [Google Scholar]
  14. Siegel V., Walter P. Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: analysis of biochemical mutants of SRP. Cell. 1988 Jan 15;52(1):39–49. doi: 10.1016/0092-8674(88)90529-6. [DOI] [PubMed] [Google Scholar]
  15. Siegel V., Walter P. Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane. J Cell Biol. 1985 Jun;100(6):1913–1921. doi: 10.1083/jcb.100.6.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Walter P., Blobel G. Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J Cell Biol. 1981 Nov;91(2 Pt 1):557–561. doi: 10.1083/jcb.91.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Walter P., Blobel G. Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):551–556. doi: 10.1083/jcb.91.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Walter P., Gilmore R., Blobel G. Protein translocation across the endoplasmic reticulum. Cell. 1984 Aug;38(1):5–8. doi: 10.1016/0092-8674(84)90520-8. [DOI] [PubMed] [Google Scholar]
  19. Walter P., Ibrahimi I., Blobel G. Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):545–550. doi: 10.1083/jcb.91.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wiedmann M., Kurzchalia T. V., Bielka H., Rapoport T. A. Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking. J Cell Biol. 1987 Feb;104(2):201–208. doi: 10.1083/jcb.104.2.201. [DOI] [PMC free article] [PubMed] [Google Scholar]

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