Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1995 Dec;109(4):1267–1276. doi: 10.1104/pp.109.4.1267

Light-harvesting chlorophyll a/b-binding protein inserted into isolated thylakoids binds pigments and is assembled into trimeric light-harvesting complex.

A Kuttkat 1, R Grimm 1, H Paulsen 1
PMCID: PMC157659  PMID: 8539291

Abstract

The light-harvesting chlorophyll a/b-binding protein (LHCP) is largely protected against protease (except for about 1 kD on the N terminus) in the thylakoid membrane; this protease resistance is often used to assay successful insertion of LHCP into isolated thylakoids in vitro. In this paper we show that this protease resistance is exhibited by trimeric light-harvesting complex of photosystem II (LHCII) but not by monomeric LHCII in which about 5 kD on the N terminus of LHCP are cleaved off by protease. When a mutant version of LHCP that is unable to trimerize in an in vitro reconstitution assay is inserted into isolated thylakoids, it gives rise to only the shorter protease digestion product indicative of monomeric LHCII. We conclude that more of the N-terminal domain of LHCP is shielded in trimeric than in monomeric LHCII and that this difference in protease sensitivity can be used to distinguish between LHCP assembled in LHCII monomers or trimers. The data presented prove that upon insertion of LHCP into isolated thylakoids at least part of the protein spontaneously binds pigments to form LHCII, which then is assembled in trimers. The dependence of the protease sensitivity of thylakoid-inserted LHCP on the oligomerization state of the newly formed LHCII justifies caution when using a protease assay to verify successful insertion of LHCP into the membrane.

Full Text

The Full Text of this article is available as a PDF (3.2 MB).

Selected References

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

  1. Allen K. D., Staehelin L. A. Resolution of 16 to 20 chlorophyll-protein complexes using a low ionic strength native green gel system. Anal Biochem. 1991 Apr;194(1):214–222. doi: 10.1016/0003-2697(91)90170-x. [DOI] [PubMed] [Google Scholar]
  2. Burke J. J., Ditto C. L., Arntzen C. J. Involvement of the light-harvesting complex in cation regulation of excitation energy distribution in chloroplasts. Arch Biochem Biophys. 1978 Apr 15;187(1):252–263. doi: 10.1016/0003-9861(78)90031-0. [DOI] [PubMed] [Google Scholar]
  3. Cammarata K. V., Schmidt G. W. In vitro reconstitution of a light-harvesting gene product: deletion mutagenesis and analyses of pigment binding. Biochemistry. 1992 Mar 17;31(10):2779–2789. doi: 10.1021/bi00125a019. [DOI] [PubMed] [Google Scholar]
  4. Cashmore A. R. Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide. Proc Natl Acad Sci U S A. 1984 May;81(10):2960–2964. doi: 10.1073/pnas.81.10.2960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chitnis P. R., Harel E., Kohorn B. D., Tobin E. M., Thornber J. P. Assembly of the precursor and processed light-harvesting chlorophyll a/b protein of Lemna into the light-harvesting complex II of barley etiochloroplasts. J Cell Biol. 1986 Mar;102(3):982–988. doi: 10.1083/jcb.102.3.982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Dreyfuss B. W., Thornber J. P. Assembly of the Light-Harvesting Complexes (LHCs) of Photosystem II (Monomeric LHC IIb Complexes Are Intermediates in the Formation of Oligomeric LHC IIb Complexes). Plant Physiol. 1994 Nov;106(3):829–839. doi: 10.1104/pp.106.3.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Franklin A. E., Hoffman N. E. Characterization of a chloroplast homologue of the 54-kDa subunit of the signal recognition particle. J Biol Chem. 1993 Oct 15;268(29):22175–22180. [PubMed] [Google Scholar]
  9. Hobe S., Förster R., Klingler J., Paulsen H. N-proximal sequence motif in light-harvesting chlorophyll a/b-binding protein is essential for the trimerization of light-harvesting chlorophyll a/b complex. Biochemistry. 1995 Aug 15;34(32):10224–10228. doi: 10.1021/bi00032a016. [DOI] [PubMed] [Google Scholar]
  10. Hobe S., Prytulla S., Kühlbrandt W., Paulsen H. Trimerization and crystallization of reconstituted light-harvesting chlorophyll a/b complex. EMBO J. 1994 Aug 1;13(15):3423–3429. doi: 10.1002/j.1460-2075.1994.tb06647.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Huang L., Adam Z., Hoffman N. E. Deletion Mutants of Chlorophyll a/b Binding Proteins Are Efficiently Imported into Chloroplasts but Do Not Integrate into Thylakoid Membranes. Plant Physiol. 1992 May;99(1):247–255. doi: 10.1104/pp.99.1.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jansson S. The light-harvesting chlorophyll a/b-binding proteins. Biochim Biophys Acta. 1994 Feb 8;1184(1):1–19. doi: 10.1016/0005-2728(94)90148-1. [DOI] [PubMed] [Google Scholar]
  13. Kohorn B. D., Auchincloss A. H. Integration of a chlorophyll-binding protein into Escherichia coli membranes in the absence of chlorophyll. J Biol Chem. 1991 Jun 25;266(18):12048–12052. [PubMed] [Google Scholar]
  14. Kohorn B. D., Harel E., Chitnis P. R., Thornber J. P., Tobin E. M. Functional and mutational analysis of the light-harvesting chlorophyll a/b protein of thylakoid membranes. J Cell Biol. 1986 Mar;102(3):972–981. doi: 10.1083/jcb.102.3.972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  16. Kühlbrandt W., Wang D. N., Fujiyoshi Y. Atomic model of plant light-harvesting complex by electron crystallography. Nature. 1994 Feb 17;367(6464):614–621. doi: 10.1038/367614a0. [DOI] [PubMed] [Google Scholar]
  17. Kühlbrandt W., Wang D. N. Three-dimensional structure of plant light-harvesting complex determined by electron crystallography. Nature. 1991 Mar 14;350(6314):130–134. doi: 10.1038/350130a0. [DOI] [PubMed] [Google Scholar]
  18. Nussberger S., Dörr K., Wang D. N., Kühlbrandt W. Lipid-protein interactions in crystals of plant light-harvesting complex. J Mol Biol. 1993 Nov 20;234(2):347–356. doi: 10.1006/jmbi.1993.1591. [DOI] [PubMed] [Google Scholar]
  19. Paulsen H., Finkenzeller B., Kühlein N. Pigments induce folding of light-harvesting chlorophyll a/b-binding protein. Eur J Biochem. 1993 Aug 1;215(3):809–816. doi: 10.1111/j.1432-1033.1993.tb18096.x. [DOI] [PubMed] [Google Scholar]
  20. Paulsen H., Hobe S. Pigment-binding properties of mutant light-harvesting chlorophyll-a/b-binding protein. Eur J Biochem. 1992 Apr 1;205(1):71–76. doi: 10.1111/j.1432-1033.1992.tb16752.x. [DOI] [PubMed] [Google Scholar]
  21. Paulsen H., Kuttkat A. Pigment complexes of light-harvesting chlorophyll a/b binding protein are stabilized by a segment in the carboxyterminal hydrophilic domain of the protein. Photochem Photobiol. 1993 Jan;57(1):139–142. doi: 10.1111/j.1751-1097.1993.tb02269.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Plumley F. G., Schmidt G. W. Reconstitution of chlorophyll a/b light-harvesting complexes: Xanthophyll-dependent assembly and energy transfer. Proc Natl Acad Sci U S A. 1987 Jan;84(1):146–150. doi: 10.1073/pnas.84.1.146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Plumley G. F., Schmidt G. W. Light-Harvesting Chlorophyll a/b Complexes: Interdependent Pigment Synthesis and Protein Assembly. Plant Cell. 1995 Jun;7(6):689–704. doi: 10.1105/tpc.7.6.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reed J. E., Cline K., Stephens L. C., Bacot K. O., Viitanen P. V. Early events in the import/assembly pathway of an integral thylakoid protein. Eur J Biochem. 1990 Nov 26;194(1):33–42. doi: 10.1111/j.1432-1033.1990.tb19423.x. [DOI] [PubMed] [Google Scholar]
  26. Schmidt G. W., Bartlett S. G., Grossman A. R., Cashmore A. R., Chua N. H. Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex. J Cell Biol. 1981 Nov;91(2 Pt 1):468–478. doi: 10.1083/jcb.91.2.468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yalovsky S., Ne'eman E., Schuster G., Paulsen H., Harel E., Nechushtai R. Accumulation of a light-harvesting chlorophyll a/b protein in the chloroplast grana lamellae. The lateral migration of the membrane protein precursor is independent of its processing. J Biol Chem. 1992 Oct 15;267(29):20689–20693. [PubMed] [Google Scholar]
  28. 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]

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

RESOURCES