Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1989 Nov;8(11):3195–3206. doi: 10.1002/j.1460-2075.1989.tb08478.x

The transit peptide of a chloroplast thylakoid membrane protein is functionally equivalent to a stromal-targeting sequence.

J M Hand 1, L J Szabo 1, A C Vasconcelos 1, A R Cashmore 1
PMCID: PMC401437  PMID: 2684639

Abstract

The role of transit peptides in intraorganellar targeting has been studied for a chlorophyll a/b binding (CAB) polypeptide of photosystem II (PSII) and the small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS) from Pisum sativum (pea). These studies have involved in vitro import of fusion proteins into isolated pea chloroplasts. Fusion of the CAB transit peptide to RBCS mediates import to the stroma, as evidenced by assembly of RBCS with chloroplast-synthesized large subunit (RBCL) to form holoenzyme. Similarly, fusion of the RBCS transit peptide to the mature CAB polypeptide mediates import and results in integration of the processed CAB protein into the thylakoid membrane. Correct integration was indicated by association with PSII and assembly with chlorophyll to form the light-harvesting chlorophyll a/b protein complex (LHCII). We interpret these results as evidence that the CAB transit peptide is functionally equivalent to a stromal-targeting sequence and that intraorganellar sorting of the CAB protein must be determined by sequences residing within the mature protein. Our results and those of others suggest that import and integration of CAB polypeptides into the thylakoid proceeds via the stroma.

Full text

PDF
3206

Images in this article

3196Image
on p.3196
3197Image
on p.3197
3198Image
on p.3198
3198Image
on p.3198
3199Image
on p.3199
3200Image
on p.3200
3201Image
on p.3201
3202Image
on p.3202

Selected References

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

  1. Arnon D. I. COPPER ENZYMES IN ISOLATED CHLOROPLASTS. POLYPHENOLOXIDASE IN BETA VULGARIS. Plant Physiol. 1949 Jan;24(1):1–15. doi: 10.1104/pp.24.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Canaani O., Barber J., Malkin S. Evidence that phosphorylation and dephosphorylation regulate the distribution of excitation energy between the two photosystems of photosynthesis in vivo: Photoacoustic and fluorimetric study of an intact leaf. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1614–1618. doi: 10.1073/pnas.81.6.1614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cashmore A. R., Broadhurst M. K., Gray R. E. Cell-free synthesis of leaf protein: Identification of an apparent precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase. Proc Natl Acad Sci U S A. 1978 Feb;75(2):655–659. doi: 10.1073/pnas.75.2.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cashmore A. R. Protein synthesis in plant leaf tissue. The sites of synthesis of the major proteins. J Biol Chem. 1976 May 10;251(9):2848–2853. [PubMed] [Google Scholar]
  7. 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]
  8. Chua N. H., Gillham N. W. The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii. J Cell Biol. 1977 Aug;74(2):441–452. doi: 10.1083/jcb.74.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chua N. H., Schmidt G. W. Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6110–6114. doi: 10.1073/pnas.75.12.6110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cline K. Import of proteins into chloroplasts. Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates. J Biol Chem. 1986 Nov 5;261(31):14804–14810. [PubMed] [Google Scholar]
  11. 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]
  12. Cline K., Werner-Washburne M., Lubben T. H., Keegstra K. Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts. J Biol Chem. 1985 Mar 25;260(6):3691–3696. [PubMed] [Google Scholar]
  13. Coruzzi G., Broglie R., Cashmore A., Chua N. H. Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide. J Biol Chem. 1983 Feb 10;258(3):1399–1402. [PubMed] [Google Scholar]
  14. Darr S. C., Somerville S. C., Arntzen C. J. Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II. J Cell Biol. 1986 Sep;103(3):733–740. doi: 10.1083/jcb.103.3.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dobberstein B., Blobel G., Chua N. H. In vitro synthesis and processing of a putative precursor for the small subunit of ribulose-1,5-bisphosphate carboxylase of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1082–1085. doi: 10.1073/pnas.74.3.1082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Douce R., Block M. A., Dorne A. J., Joyard J. The plastid envelope membranes: their structure, composition, and role in chloroplast biogenesis. Subcell Biochem. 1984;10:1–84. doi: 10.1007/978-1-4613-2709-7_1. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Flügge U. I., Hinz G. Energy dependence of protein translocation into chloroplasts. Eur J Biochem. 1986 Nov 3;160(3):563–570. doi: 10.1111/j.1432-1033.1986.tb10075.x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Guo L. H., Wu R. New rapid methods for DNA sequencing based in exonuclease III digestion followed by repair synthesis. Nucleic Acids Res. 1982 Mar 25;10(6):2065–2084. doi: 10.1093/nar/10.6.2065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Karlin-Neumann G. A., Kohorn B. D., Thornber J. P., Tobin E. M. A chlorophyll a/b-protein encoded by a gene containing an intron with characteristics of a transposable element. J Mol Appl Genet. 1985;3(1):45–61. [PubMed] [Google Scholar]
  22. Kavanagh T. A., Jefferson R. A., Bevan M. W. Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein. Mol Gen Genet. 1988 Dec;215(1):38–45. doi: 10.1007/BF00331300. [DOI] [PubMed] [Google Scholar]
  23. Kirwin P. M., Elderfield P. D., Robinson C. Transport of proteins into chloroplasts. Partial purification of a thylakoidal processing peptidase involved in plastocyanin biogenesis. J Biol Chem. 1987 Dec 5;262(34):16386–16390. [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Kohorn B. D., Tobin E. M. A hydrophobic, carboxy-proximal region of a light-harvesting chlorophyll a/b protein is necessary for stable integration into thylakoid membranes. Plant Cell. 1989 Jan;1(1):159–166. doi: 10.1105/tpc.1.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lamppa G. K., Abad M. S. Processing of a wheat light-harvesting chlorophyll a/b protein precursor by a soluble enzyme from higher plant chloroplasts. J Cell Biol. 1987 Dec;105(6 Pt 1):2641–2648. doi: 10.1083/jcb.105.6.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lamppa G. K. The chlorophyll a/b-binding protein inserts into the thylakoids independent of its cognate transit peptide. J Biol Chem. 1988 Oct 15;263(29):14996–14999. [PubMed] [Google Scholar]
  29. Lubben T. H., Keegstra K. Efficient in vitro import of a cytosolic heat shock protein into pea chloroplasts. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5502–5506. doi: 10.1073/pnas.83.15.5502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mishkind M. L., Wessler S. R., Schmidt G. W. Functional determinants in transit sequences: import and partial maturation by vascular plant chloroplasts of the ribulose-1,5-bisphosphate carboxylase small subunit of Chlamydomonas. J Cell Biol. 1985 Jan;100(1):226–234. doi: 10.1083/jcb.100.1.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mullet J. E. The amino acid sequence of the polypeptide segment which regulates membrane adhesion (grana stacking) in chloroplasts. J Biol Chem. 1983 Aug 25;258(16):9941–9948. [PubMed] [Google Scholar]
  32. Pain D., Blobel G. Protein import into chloroplasts requires a chloroplast ATPase. Proc Natl Acad Sci U S A. 1987 May;84(10):3288–3292. doi: 10.1073/pnas.84.10.3288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pain D., Kanwar Y. S., Blobel G. Identification of a receptor for protein import into chloroplasts and its localization to envelope contact zones. Nature. 1988 Jan 21;331(6153):232–237. doi: 10.1038/331232a0. [DOI] [PubMed] [Google Scholar]
  34. Robinson C., Ellis R. J. Transport of proteins into chloroplasts. Partial purification of a chloroplast protease involved in the processing of important precursor polypeptides. Eur J Biochem. 1984 Jul 16;142(2):337–342. doi: 10.1111/j.1432-1033.1984.tb08291.x. [DOI] [PubMed] [Google Scholar]
  35. Robinson C., Ellis R. J. Transport of proteins into chloroplasts. The precursor of small subunit of ribulose bisphosphate carboxylase is processed to the mature size in two steps. Eur J Biochem. 1984 Jul 16;142(2):343–346. doi: 10.1111/j.1432-1033.1984.tb08292.x. [DOI] [PubMed] [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Smeekens S., Bauerle C., Hageman J., Keegstra K., Weisbeek P. The role of the transit peptide in the routing of precursors toward different chloroplast compartments. Cell. 1986 Aug 1;46(3):365–375. doi: 10.1016/0092-8674(86)90657-4. [DOI] [PubMed] [Google Scholar]
  39. Van den Broeck G., Timko M. P., Kausch A. P., Cashmore A. R., Van Montagu M., Herrera-Estrella L. Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase. 1985 Jan 31-Feb 6Nature. 313(6001):358–363. doi: 10.1038/313358a0. [DOI] [PubMed] [Google Scholar]
  40. Viitanen P. V., Doran E. R., Dunsmuir P. What is the role of the transit peptide in thylakoid integration of the light-harvesting chlorophyll a/b protein? J Biol Chem. 1988 Oct 15;263(29):15000–15007. [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES