Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Nov 2;123(4):799–807. doi: 10.1083/jcb.123.4.799

GTP hydrolysis by complexes of the signal recognition particle and the signal recognition particle receptor

PMCID: PMC2200155  PMID: 8227141

Abstract

Translocation of proteins across the endoplasmic reticulum membrane is a GTP-dependent process. The signal recognition particle (SRP) and the SRP receptor both contain subunits with GTP binding domains. One GTP- dependent reaction during protein translocation is the SRP receptor- mediated dissociation of SRP from the signal sequence of a nascent polypeptide. Here, we have assayed the SRP and the SRP receptor for GTP binding and hydrolysis activities. GTP hydrolysis by SRP was not detected, so the maximal GTP hydrolysis rate for SRP was estimated to be < 0.002 mol GTP hydrolyzed x mol of SRP-1 x min-1. The intrinsic GTP hydrolysis activity of the SRP receptor ranged between 0.02 and 0.04 mol GTP hydrolyzed x mol of SRP receptor-1 x min-1. A 40-fold enhancement of GTP hydrolysis activity relative to that observed for the SRP receptor alone was obtained when complexes were formed between SRP and the SRP receptor. GTP hydrolysis activity was inhibited by GDP, but not by ATP. Extended incubation of the SRP or the SRP receptor with GTP resulted in substoichiometric quantities of protein-bound ribonucleotide. SRP-SRP receptor complexes engaged in GTP hydrolysis were found to contain a minimum of one bound guanine ribonucleotide per SRP-SRP receptor complex. We conclude that the GTP hydrolysis activity described here is indicative of one of the GTPase cycles that occur during protein translocation across the endoplasmic reticulum.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

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

  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. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  3. Brandt D. R., Asano T., Pedersen S. E., Ross E. M. Reconstitution of catecholamine-stimulated guanosinetriphosphatase activity. Biochemistry. 1983 Sep 13;22(19):4357–4362. doi: 10.1021/bi00288a002. [DOI] [PubMed] [Google Scholar]
  4. Brandt D. R., Ross E. M. Catecholamine-stimulated GTPase cycle. Multiple sites of regulation by beta-adrenergic receptor and Mg2+ studied in reconstituted receptor-Gs vesicles. J Biol Chem. 1986 Feb 5;261(4):1656–1664. [PubMed] [Google Scholar]
  5. Brandt D. R., Ross E. M. GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates. J Biol Chem. 1985 Jan 10;260(1):266–272. [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Connolly T., Rapiejko P. J., Gilmore R. Requirement of GTP hydrolysis for dissociation of the signal recognition particle from its receptor. Science. 1991 May 24;252(5009):1171–1173. doi: 10.1126/science.252.5009.1171. [DOI] [PubMed] [Google Scholar]
  9. Der C. J., Finkel T., Cooper G. M. Biological and biochemical properties of human rasH genes mutated at codon 61. Cell. 1986 Jan 17;44(1):167–176. doi: 10.1016/0092-8674(86)90495-2. [DOI] [PubMed] [Google Scholar]
  10. Dever T. E., Glynias M. J., Merrick W. C. GTP-binding domain: three consensus sequence elements with distinct spacing. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1814–1818. doi: 10.1073/pnas.84.7.1814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibbs J. B., Sigal I. S., Poe M., Scolnick E. M. Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5704–5708. doi: 10.1073/pnas.81.18.5704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Görlich D., Prehn S., Hartmann E., Kalies K. U., Rapoport T. A. A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation. Cell. 1992 Oct 30;71(3):489–503. doi: 10.1016/0092-8674(92)90517-g. [DOI] [PubMed] [Google Scholar]
  16. High S., Flint N., Dobberstein B. Requirements for the membrane insertion of signal-anchor type proteins. J Cell Biol. 1991 Apr;113(1):25–34. doi: 10.1083/jcb.113.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hoffman K. E., Gilmore R. Guanosine triphosphate promotes the post-translational integration of opsin into the endoplasmic reticulum membrane. J Biol Chem. 1988 Mar 25;263(9):4381–4385. [PubMed] [Google Scholar]
  18. Hortsch M., Labeit S., Meyer D. I. Complete cDNA sequence coding for human docking protein. Nucleic Acids Res. 1988 Jan 11;16(1):361–362. doi: 10.1093/nar/16.1.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kabcenell A. K., Goud B., Northup J. K., Novick P. J. Binding and hydrolysis of guanine nucleotides by Sec4p, a yeast protein involved in the regulation of vesicular traffic. J Biol Chem. 1990 Jun 5;265(16):9366–9372. [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Lauffer L., Garcia P. D., Harkins R. N., Coussens L., Ullrich A., Walter P. Topology of signal recognition particle receptor in endoplasmic reticulum membrane. 1985 Nov 28-Dec 4Nature. 318(6044):334–338. doi: 10.1038/318334a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Milburn M. V., Tong L., deVos A. M., Brünger A., Yamaizumi Z., Nishimura S., Kim S. H. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science. 1990 Feb 23;247(4945):939–945. doi: 10.1126/science.2406906. [DOI] [PubMed] [Google Scholar]
  25. Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Rapiejko P. J., Gilmore R. Protein translocation across the ER requires a functional GTP binding site in the alpha subunit of the signal recognition particle receptor. J Cell Biol. 1992 May;117(3):493–503. doi: 10.1083/jcb.117.3.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Siegel V., Walter P. Binding sites of the 19-kDa and 68/72-kDa signal recognition particle (SRP) proteins on SRP RNA as determined in protein-RNA "footprinting". Proc Natl Acad Sci U S A. 1988 Mar;85(6):1801–1805. doi: 10.1073/pnas.85.6.1801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Simon S. M., Blobel G. A protein-conducting channel in the endoplasmic reticulum. Cell. 1991 May 3;65(3):371–380. doi: 10.1016/0092-8674(91)90455-8. [DOI] [PubMed] [Google Scholar]
  33. Simon S. M., Blobel G. Signal peptides open protein-conducting channels in E. coli. Cell. 1992 May 15;69(4):677–684. doi: 10.1016/0092-8674(92)90231-z. [DOI] [PubMed] [Google Scholar]
  34. Sunyer T., Codina J., Birnbaumer L. GTP hydrolysis by pure Ni, the inhibitory regulatory component of adenylyl cyclases. J Biol Chem. 1984 Dec 25;259(24):15447–15451. [PubMed] [Google Scholar]
  35. 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]
  36. Tamir A., Fawzi A. B., Northup J. K. Unique guanine nucleotide binding properties of the human placental GTP-binding protein Gp. Biochemistry. 1990 Jul 31;29(30):6947–6954. doi: 10.1021/bi00482a002. [DOI] [PubMed] [Google Scholar]
  37. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  38. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Walter P., Blobel G. Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7112–7116. doi: 10.1073/pnas.77.12.7112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. 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]
  44. 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]
  45. Zopf D., Bernstein H. D., Walter P. GTPase domain of the 54-kD subunit of the mammalian signal recognition particle is required for protein translocation but not for signal sequence binding. J Cell Biol. 1993 Mar;120(5):1113–1121. doi: 10.1083/jcb.120.5.1113. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES