Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1994 May;105(1):295–304. doi: 10.1104/pp.105.1.295

Evidence for a stromal GTP requirement for the integration of a chlorophyll a/b-binding polypeptide into thylakoid membranes.

N E Hoffman 1, A E Franklin 1
PMCID: PMC159357  PMID: 8029355

Abstract

The integration of chlorophyll a/b-binding (LHCP) polypeptides and the translocation of the 33-kD oxygen-evolving enhancer protein (OEE33) have been previously shown to occur in chloroplast extracts containing stroma, thylakoids, ATP, and MgCl2. We have re-examined the nucleotide requirement for these two reactions using stromal extract and translation products depleted of low molecular weight compounds. LHCP integration activity was up to 10-fold higher when assayed with GTP compared with ATP, CTP, or UTP. A combination of ATP and GTP supported less LHCP integration activity than GTP alone, suggesting that GTP meets the entire nucleotide requirement. Nonhydrolyzable analogs of GTP were inhibitory, consistent with the idea that GTP hydrolysis is required for integration activity. Periodate-oxidized GTP (GTPox) also inhibited the integration reaction when present during the assay. Pretreatment of stroma with GTPox followed by GTPox removal inhibited integration activity, whereas pretreatment of thylakoids had no effect. We interpret this to mean that a GTP-binding protein involved in integration is localized in the stroma. Translocation of OEE33 was more efficient with ATP than with GTP, and the combination of both nucleotides was not additive. Our data implicate the involvement of a GTPase in LHCP integration but not in the translocation of OEE33.

Full Text

The Full Text of this article is available as a PDF (3.7 MB).

Selected References

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

  1. Adam Z., Hoffman N. E. Biogenesis of a photosystem I light-harvesting complex. Evidence for a membrane intermediate. Plant Physiol. 1993 May;102(1):35–43. doi: 10.1104/pp.102.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauerle C., Dorl J., Keegstra K. Kinetic analysis of the transport of thylakoid lumenal proteins in experiments using intact chloroplasts. J Biol Chem. 1991 Mar 25;266(9):5884–5890. [PubMed] [Google Scholar]
  3. Cline K., Ettinger W. F., Theg S. M. Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP. J Biol Chem. 1992 Feb 5;267(4):2688–2696. [PubMed] [Google Scholar]
  4. Cline K., Fulsom D. R., Viitanen P. V. An imported thylakoid protein accumulates in the stroma when insertion into thylakoids is inhibited. J Biol Chem. 1989 Aug 25;264(24):14225–14232. [PubMed] [Google Scholar]
  5. Cline K. Light-Harvesting Chlorophyll a/b Protein : Membrane Insertion, Proteolytic Processing, Assembly into LHC II, and Localization to Appressed Membranes Occurs in Chloroplast Lysates. Plant Physiol. 1988 Apr;86(4):1120–1126. doi: 10.1104/pp.86.4.1120. [DOI] [PMC free article] [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. Flaherty K. M., DeLuca-Flaherty C., McKay D. B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature. 1990 Aug 16;346(6285):623–628. doi: 10.1038/346623a0. [DOI] [PubMed] [Google Scholar]
  10. Flynn G. C., Chappell T. G., Rothman J. E. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science. 1989 Jul 28;245(4916):385–390. doi: 10.1126/science.2756425. [DOI] [PubMed] [Google Scholar]
  11. Fulson D. R., Cline K. A Soluble Protein Factor is Required in Vitro for Membrane Insertion of the Thylakoid Precursor Protein, pLHCP. Plant Physiol. 1988 Dec;88(4):1146–1153. doi: 10.1104/pp.88.4.1146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gilmore A. M., Yamamoto H. Y. Dark induction of zeaxanthin-dependent nonphotochemical fluorescence quenching mediated by ATP. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1899–1903. doi: 10.1073/pnas.89.5.1899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hingorani V. N., Chang L. F., Ho Y. K. Chemical modification of bovine transducin: probing the GTP-binding site with affinity analogues. Biochemistry. 1989 Sep 5;28(18):7424–7432. doi: 10.1021/bi00444a041. [DOI] [PubMed] [Google Scholar]
  14. Klösgen R. B., Brock I. W., Herrmann R. G., Robinson C. Proton gradient-driven import of the 16 kDa oxygen-evolving complex protein as the full precursor protein by isolated thylakoids. Plant Mol Biol. 1992 Mar;18(5):1031–1034. doi: 10.1007/BF00019226. [DOI] [PubMed] [Google Scholar]
  15. Ko K., Cashmore A. R. Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein. EMBO J. 1989 Nov;8(11):3187–3194. doi: 10.1002/j.1460-2075.1989.tb08477.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Luirink J., High S., Wood H., Giner A., Tollervey D., Dobberstein B. Signal-sequence recognition by an Escherichia coli ribonucleoprotein complex. Nature. 1992 Oct 22;359(6397):741–743. doi: 10.1038/359741a0. [DOI] [PubMed] [Google Scholar]
  17. Mould R. M., Robinson C. A proton gradient is required for the transport of two lumenal oxygen-evolving proteins across the thylakoid membrane. J Biol Chem. 1991 Jul 5;266(19):12189–12193. [PubMed] [Google Scholar]
  18. Mould R. M., Shackleton J. B., Robinson C. Transport of proteins into chloroplasts. Requirements for the efficient import of two lumenal oxygen-evolving complex proteins into isolated thylakoids. J Biol Chem. 1991 Sep 15;266(26):17286–17289. [PubMed] [Google Scholar]
  19. Mould R. M., Shackleton J. B., Robinson C. Transport of proteins into chloroplasts. Requirements for the efficient import of two lumenal oxygen-evolving complex proteins into isolated thylakoids. J Biol Chem. 1991 Sep 15;266(26):17286–17289. [PubMed] [Google Scholar]
  20. Neupert W., Pfanner N. Roles of molecular chaperones in protein targeting to mitochondria. Philos Trans R Soc Lond B Biol Sci. 1993 Mar 29;339(1289):355–362. doi: 10.1098/rstb.1993.0034. [DOI] [PubMed] [Google Scholar]
  21. Payan L. A., Cline K. A stromal protein factor maintains the solubility and insertion competence of an imported thylakoid membrane protein. J Cell Biol. 1991 Feb;112(4):603–613. doi: 10.1083/jcb.112.4.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Ribes V., Römisch K., Giner A., Dobberstein B., Tollervey D. E. coli 4.5S RNA is part of a ribonucleoprotein particle that has properties related to signal recognition particle. Cell. 1990 Nov 2;63(3):591–600. doi: 10.1016/0092-8674(90)90454-m. [DOI] [PubMed] [Google Scholar]
  24. Robinson C., Klösgen R. B., Herrmann R. G., Shackleton J. B. Protein translocation across the thylakoid membrane--a tale of two mechanisms. FEBS Lett. 1993 Jun 28;325(1-2):67–69. doi: 10.1016/0014-5793(93)81415-v. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Wilson C., Connolly T., Morrison T., Gilmore R. Integration of membrane proteins into the endoplasmic reticulum requires GTP. J Cell Biol. 1988 Jul;107(1):69–77. doi: 10.1083/jcb.107.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yuan J., Henry R., Cline K. Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein Hsp70. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8552–8556. doi: 10.1073/pnas.90.18.8552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. von Heijne G., Steppuhn J., Herrmann R. G. Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem. 1989 Apr 1;180(3):535–545. doi: 10.1111/j.1432-1033.1989.tb14679.x. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES