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. 1993 Mar 1;120(5):1113–1121. doi: 10.1083/jcb.120.5.1113

GTPase domain of the 54-kD subunit of the mammalian signal recognition particle is required for protein translocation but not for signal sequence binding

PMCID: PMC2119734  PMID: 8382204

Abstract

The 54-kD subunit of the signal recognition particle (SRP54) binds to signal sequences of nascent secretory and transmembrane proteins. SRP54 consists of two separable domains, a 33-kD amino-terminal domain that contains a GTP-binding site (SRP54G) and a 22-kD carboxy-terminal domain (SRP54M) containing binding sites for both the signal sequence and SRP RNA. To examine the function of the two domains in more detail, we have purified SRP54M and used it to assemble a partial SRP that lacks the amino-terminal domain of SRP54 [SRP(-54G)]. This particle recognized signal sequences in two independent assays, albeit less efficiently than intact SRP. Analysis of the signal sequence binding activity of free SRP54 and SRP54M supports the conclusion that SRP54M binds signal sequences with lower affinity than the intact protein. In contrast, when SRP(-54G) was assayed for its ability to promote the translocation of preprolactin across microsomal membranes, it was completely inactive, apparently because it was unable to interact normally with the SRP receptor. These results imply that SRP54G plays an essential role in SRP-mediated targeting of nascent chain-ribosome complexes to the ER membrane and also influences signal sequence recognition, possibly by promoting a tighter association between signal sequences and SRP54M.

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Selected References

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  1. Bernstein H. D., Poritz M. A., Strub K., Hoben P. J., Brenner S., Walter P. Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle. Nature. 1989 Aug 10;340(6233):482–486. doi: 10.1038/340482a0. [DOI] [PubMed] [Google Scholar]
  2. Blobel G., Sabatini D. Dissociation of mammalian polyribosomes into subunits by puromycin. Proc Natl Acad Sci U S A. 1971 Feb;68(2):390–394. doi: 10.1073/pnas.68.2.390. [DOI] [PMC free article] [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. Connolly T., Gilmore R. The signal recognition particle receptor mediates the GTP-dependent displacement of SRP from the signal sequence of the nascent polypeptide. Cell. 1989 May 19;57(4):599–610. doi: 10.1016/0092-8674(89)90129-3. [DOI] [PubMed] [Google Scholar]
  5. Gilmore R., Blobel G., Walter P. Protein translocation across the endoplasmic reticulum. I. Detection in the microsomal membrane of a receptor for the signal recognition particle. J Cell Biol. 1982 Nov;95(2 Pt 1):463–469. doi: 10.1083/jcb.95.2.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. High S., Dobberstein B. The signal sequence interacts with the methionine-rich domain of the 54-kD protein of signal recognition particle. J Cell Biol. 1991 Apr;113(2):229–233. doi: 10.1083/jcb.113.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Krieg U. C., Walter P., Johnson A. E. Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8604–8608. doi: 10.1073/pnas.83.22.8604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kurzchalia T. V., Wiedmann M., Girshovich A. S., Bochkareva E. S., Bielka H., Rapoport T. A. The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle. Nature. 1986 Apr 17;320(6063):634–636. doi: 10.1038/320634a0. [DOI] [PubMed] [Google Scholar]
  11. Lütcke H., High S., Römisch K., Ashford A. J., Dobberstein B. The methionine-rich domain of the 54 kDa subunit of signal recognition particle is sufficient for the interaction with signal sequences. EMBO J. 1992 Apr;11(4):1543–1551. doi: 10.1002/j.1460-2075.1992.tb05199.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Migliaccio G., Nicchitta C. V., Blobel G. The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation. J Cell Biol. 1992 Apr;117(1):15–25. doi: 10.1083/jcb.117.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Poritz M. A., Bernstein H. D., Strub K., Zopf D., Wilhelm H., Walter P. An E. coli ribonucleoprotein containing 4.5S RNA resembles mammalian signal recognition particle. Science. 1990 Nov 23;250(4984):1111–1117. doi: 10.1126/science.1701272. [DOI] [PubMed] [Google Scholar]
  15. Römisch K., Webb J., Herz J., Prehn S., Frank R., Vingron M., Dobberstein B. Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains. Nature. 1989 Aug 10;340(6233):478–482. doi: 10.1038/340478a0. [DOI] [PubMed] [Google Scholar]
  16. Römisch K., Webb J., Lingelbach K., Gausepohl H., Dobberstein B. The 54-kD protein of signal recognition particle contains a methionine-rich RNA binding domain. J Cell Biol. 1990 Nov;111(5 Pt 1):1793–1802. doi: 10.1083/jcb.111.5.1793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Siegel V., Walter P. Removal of the Alu structural domain from signal recognition particle leaves its protein translocation activity intact. Nature. 1986 Mar 6;320(6057):81–84. doi: 10.1038/320081a0. [DOI] [PubMed] [Google Scholar]
  20. Strub K., Walter P. Assembly of the Alu domain of the signal recognition particle (SRP): dimerization of the two protein components is required for efficient binding to SRP RNA. Mol Cell Biol. 1990 Feb;10(2):777–784. doi: 10.1128/mcb.10.2.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tajima S., Lauffer L., Rath V. L., Walter P. The signal recognition particle receptor is a complex that contains two distinct polypeptide chains. J Cell Biol. 1986 Oct;103(4):1167–1178. doi: 10.1083/jcb.103.4.1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Walter P., Blobel G. Disassembly and reconstitution of signal recognition particle. Cell. 1983 Sep;34(2):525–533. doi: 10.1016/0092-8674(83)90385-9. [DOI] [PubMed] [Google Scholar]
  23. Walter P., Blobel G. Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature. 1982 Oct 21;299(5885):691–698. doi: 10.1038/299691a0. [DOI] [PubMed] [Google Scholar]
  24. Walter P., Blobel G. Signal recognition particle: a ribonucleoprotein required for cotranslational translocation of proteins, isolation and properties. Methods Enzymol. 1983;96:682–691. doi: 10.1016/s0076-6879(83)96057-3. [DOI] [PubMed] [Google Scholar]
  25. Walter P., Blobel G. Subcellular distribution of signal recognition particle and 7SL-RNA determined with polypeptide-specific antibodies and complementary DNA probe. J Cell Biol. 1983 Dec;97(6):1693–1699. doi: 10.1083/jcb.97.6.1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Walter P., Lingappa V. R. Mechanism of protein translocation across the endoplasmic reticulum membrane. Annu Rev Cell Biol. 1986;2:499–516. doi: 10.1146/annurev.cb.02.110186.002435. [DOI] [PubMed] [Google Scholar]
  28. Wolin S. L., Walter P. Signal recognition particle mediates a transient elongation arrest of preprolactin in reticulocyte lysate. J Cell Biol. 1989 Dec;109(6 Pt 1):2617–2622. doi: 10.1083/jcb.109.6.2617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zopf D., Bernstein H. D., Johnson A. E., Walter P. The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence. EMBO J. 1990 Dec;9(13):4511–4517. doi: 10.1002/j.1460-2075.1990.tb07902.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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