Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1996 Jun 25;93(13):6338–6342. doi: 10.1073/pnas.93.13.6338

The precursor of PsaD assembles into the photosystem I complex in two steps.

L Minai 1, Y Cohen 1, P R Chitnis 1, R Nechushtai 1
PMCID: PMC39023  PMID: 8692816

Abstract

The present study addresses the assembly in the chloroplast thylakoid membranes of PsaD, a peripheral membrane protein of the photosystem I complex. Located on the stromal side of the thylakoids, PsaD was found to assemble in vitro into the membranes in its precursor (pre-PsaD) and also in its mature (PsaD) form. Newly assembled unprocessed pre-PsaD was resistant to NaBr and alkaline wash. Yet it was sensitive to proteolytic digestion. In contradistinction, when the assembled precursor was processed, the resulting mature PsaD was resistant to proteases to the same extent as endogenous [correction of endogeneous] PsaD. The accumulation of protease-resistant PsaD in the thylakoids correlated with the increase of mature-PsaD in the membranes. This protection of mature PsaD from proteolysis could not be observed when PsaD was in a soluble form-i.e. not assembled within the thylakoids. The data suggest that pre-PsaD assembles to the membranes and only in a second step processing takes place. The observation that the assembly of pre-PsaD is affected by salts to a much lesser extent than that of mature-PsaD supports a two-step assembly of pre-PsaD.

Full text

PDF
6338

Images in this article

6339Fig. 1
on p.6339
6340Fig. 2
on p.6340
6340Fig. 4
on p.6340
6341Fig. 5
on p.6341

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. 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]
  3. Avni A., Avital S., Gromet-Elhanan Z. Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha. J Biol Chem. 1991 Apr 25;266(12):7317–7320. [PubMed] [Google Scholar]
  4. Baker K. P., Schaniel A., Vestweber D., Schatz G. A yeast mitochondrial outer membrane protein essential for protein import and cell viability. Nature. 1990 Dec 13;348(6302):605–609. doi: 10.1038/348605a0. [DOI] [PubMed] [Google Scholar]
  5. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Chitnis P. R., Nelson N. Assembly of Two Subunits of the Cyanobacterial Photosystem I on the n-Side of Thylakoid Membranes. Plant Physiol. 1992 May;99(1):239–246. doi: 10.1104/pp.99.1.239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chitnis P. R., Reilly P. A., Miedel M. C., Nelson N. Structure and targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem. 1989 Nov 5;264(31):18374–18380. [PubMed] [Google Scholar]
  9. 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]
  10. Cohen Y., Chitnis V. P., Nechushtai R., Chitnis P. R. Stable assembly of PsaE into cyanobacterial photosynthetic membranes is dependent on the presence of other accessory subunits of photosystem I. Plant Mol Biol. 1993 Nov;23(4):895–900. doi: 10.1007/BF00021544. [DOI] [PubMed] [Google Scholar]
  11. Cohen Y., Nechushtai R. Assembly and processing of subunit II (PsaD) precursor in the isolated photosystem-I complex. FEBS Lett. 1992 May 4;302(1):15–17. doi: 10.1016/0014-5793(92)80273-j. [DOI] [PubMed] [Google Scholar]
  12. Cohen Y., Steppuhn J., Herrmann R. G., Yalovsky S., Nechushtai R. Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing. EMBO J. 1992 Jan;11(1):79–85. doi: 10.1002/j.1460-2075.1992.tb05030.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cohen Y., Yalovsky S., Nechushtai R. Integration and assembly of photosynthetic protein complexes in chloroplast thylakoid membranes. Biochim Biophys Acta. 1995 May 8;1241(1):1–30. doi: 10.1016/0304-4157(94)00012-3. [DOI] [PubMed] [Google Scholar]
  14. Douwe de Boer A., Weisbeek P. J. Chloroplast protein topogenesis: import, sorting and assembly. Biochim Biophys Acta. 1991 Nov 13;1071(3):221–253. doi: 10.1016/0304-4157(91)90015-o. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. He W. Z., Malkin R. Specific release of a 9-kDa extrinsic polypeptide of photosystem I from spinach chloroplasts by salt washing. FEBS Lett. 1992 Aug 24;308(3):298–300. doi: 10.1016/0014-5793(92)81297-y. [DOI] [PubMed] [Google Scholar]
  17. Hoffman N. E., Franklin A. E. Evidence for a stromal GTP requirement for the integration of a chlorophyll a/b-binding polypeptide into thylakoid membranes. Plant Physiol. 1994 May;105(1):295–304. doi: 10.1104/pp.105.1.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hulford A., Hazell L., Mould R. M., Robinson C. Two distinct mechanisms for the translocation of proteins across the thylakoid membrane, one requiring the presence of a stromal protein factor and nucleotide triphosphates. J Biol Chem. 1994 Feb 4;269(5):3251–3256. [PubMed] [Google Scholar]
  19. Kassenbrock C. K., Garcia P. D., Walter P., Kelly R. B. Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature. 1988 May 5;333(6168):90–93. doi: 10.1038/333090a0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. Lagoutte B. Cloning and sequencing of spinach cDNA clones encoding the 20 kDa PS I polypeptide. FEBS Lett. 1988 May 23;232(2):275–280. doi: 10.1016/0014-5793(88)80752-x. [DOI] [PubMed] [Google Scholar]
  24. Lagoutte B., Vallon O. Purification and membrane topology of PSI-D and PSI-E, two subunits of the photosystem I reaction center. Eur J Biochem. 1992 May 1;205(3):1175–1185. doi: 10.1111/j.1432-1033.1992.tb16888.x. [DOI] [PubMed] [Google Scholar]
  25. Li N., Warren P. V., Golbeck J. H., Frank G., Zuber H., Bryant D. A. Polypeptide composition of the Photosystem I complex and the Photosystem I core protein from Synechococcus sp. PCC 6301. Biochim Biophys Acta. 1991 Aug 23;1059(2):215–225. doi: 10.1016/s0005-2728(05)80206-3. [DOI] [PubMed] [Google Scholar]
  26. Li N., Zhao J. D., Warren P. V., Warden J. T., Bryant D. A., Golbeck J. H. PsaD is required for the stable binding of PsaC to the photosystem I core protein of Synechococcus sp. PCC 6301. Biochemistry. 1991 Aug 6;30(31):7863–7872. doi: 10.1021/bi00245a028. [DOI] [PubMed] [Google Scholar]
  27. Li X., Henry R., Yuan J., Cline K., Hoffman N. E. A chloroplast homologue of the signal recognition particle subunit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3789–3793. doi: 10.1073/pnas.92.9.3789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lotan O., Cohen Y., Michaeli D., Nechushtai R. High levels of photosystem I subunit II (PsaD) mRNA result in the accumulation of the PsaD polypeptide only in the presence of light. J Biol Chem. 1993 Aug 5;268(22):16185–16189. [PubMed] [Google Scholar]
  29. Lubben T. H., Donaldson G. K., Viitanen P. V., Gatenby A. A. Several proteins imported into chloroplasts form stable complexes with the GroEL-related chloroplast molecular chaperone. Plant Cell. 1989 Dec;1(12):1223–1230. doi: 10.1105/tpc.1.12.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Martinez S. E., Huang D., Szczepaniak A., Cramer W. A., Smith J. L. Crystal structure of chloroplast cytochrome f reveals a novel cytochrome fold and unexpected heme ligation. Structure. 1994 Feb 15;2(2):95–105. doi: 10.1016/s0969-2126(00)00012-5. [DOI] [PubMed] [Google Scholar]
  31. Münch S., Ljungberg U., Steppuhn J., Schneiderbauer A., Nechushtai R., Beyreuther K., Herrmann R. G. Nucleotide sequences of cDNAs encoding the entire precursor polypeptides for subunits II and III of the photosystem I reaction center from spinach. Curr Genet. 1988 Nov;14(5):511–518. doi: 10.1007/BF00521277. [DOI] [PubMed] [Google Scholar]
  32. Ostermann J., Voos W., Kang P. J., Craig E. A., Neupert W., Pfanner N. Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix. FEBS Lett. 1990 Dec 17;277(1-2):281–284. doi: 10.1016/0014-5793(90)80865-g. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Randall L. L., Topping T. B., Hardy S. J. No specific recognition of leader peptide by SecB, a chaperone involved in protein export. Science. 1990 May 18;248(4957):860–863. doi: 10.1126/science.2188362. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Scherer P. E., Krieg U. C., Hwang S. T., Vestweber D., Schatz G. A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. EMBO J. 1990 Dec;9(13):4315–4322. doi: 10.1002/j.1460-2075.1990.tb07880.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  38. Sheffield W. P., Shore G. C., Randall S. K. Mitochondrial precursor protein. Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation, and import competence. J Biol Chem. 1990 Jul 5;265(19):11069–11076. [PubMed] [Google Scholar]
  39. Teschke C. M., Kim J., Song T., Park S., Park C., Randall L. L. Mutations that affect the folding of ribose-binding protein selected as suppressors of a defect in export in Escherichia coli. J Biol Chem. 1991 Jun 25;266(18):11789–11796. [PubMed] [Google Scholar]
  40. Yalovsky S., Paulsen H., Michaeli D., Chitnis P. R., Nechushtai R. Involvement of a chloroplast HSP70 heat shock protein in the integration of a protein (light-harvesting complex protein precursor) into the thylakoid membrane. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5616–5619. doi: 10.1073/pnas.89.12.5616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Yuan J., Henry R., McCaffery M., Cline K. SecA homolog in protein transport within chloroplasts: evidence for endosymbiont-derived sorting. Science. 1994 Nov 4;266(5186):796–798. doi: 10.1126/science.7973633. [DOI] [PubMed] [Google Scholar]
  43. Zanetti G., Merati G. Interaction between photosystem I and ferredoxin. Identification by chemical cross-linking of the polypeptide which binds ferredoxin. Eur J Biochem. 1987 Nov 16;169(1):143–146. doi: 10.1111/j.1432-1033.1987.tb13591.x. [DOI] [PubMed] [Google Scholar]
  44. Zilber A. L., Malkin R. Ferredoxin Cross-Links to a 22 kD Subunit of Photosystem I. Plant Physiol. 1988 Nov;88(3):810–814. doi: 10.1104/pp.88.3.810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zilber A. L., Malkin R. Organization and topology of photosystem I subunits. Plant Physiol. 1992 Jul;99(3):901–911. doi: 10.1104/pp.99.3.901. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES