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. 1995 Jun 15;14(12):2715–2722. doi: 10.1002/j.1460-2075.1995.tb07272.x

A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the delta pH-dependent thylakoidal protein translocase.

A M Chaddock 1, A Mant 1, I Karnauchov 1, S Brink 1, R G Herrmann 1, R B Klösgen 1, C Robinson 1
PMCID: PMC398390  PMID: 7796800

Abstract

The delta pH-driven and Sec-related thylakoidal protein translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH-dependent system contain a common twin-arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH-driven translocation process; substitution of the arg-arg by gln-gln or even arg-lys totally blocks translocation across the thylakoid membrane, and replacement by lys-arg reduces the rate of translocation by > 100-fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin-arg motif into a Sec-dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec-type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane protein does not affect the translocation pathway.

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Selected References

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

  1. Akita M., Sasaki S., Matsuyama S., Mizushima S. SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli. J Biol Chem. 1990 May 15;265(14):8164–8169. [PubMed] [Google Scholar]
  2. Bassham D. C., Bartling D., Mould R. M., Dunbar B., Weisbeek P., Herrmann R. G., Robinson C. Transport of proteins into chloroplasts. Delineation of envelope "transit" and thylakoid "transfer" signals within the pre-sequences of three imported thylakoid lumen proteins. J Biol Chem. 1991 Dec 15;266(35):23606–23610. [PubMed] [Google Scholar]
  3. Bovy A., de Vrieze G., Borrias M., Weisbeek P. Isolation and sequence analysis of a gene encoding a basic cytochrome c-553 from the cyanobacterium Anabaena SP. PCC 7937. Plant Mol Biol. 1992 Jun;19(3):491–492. doi: 10.1007/BF00023397. [DOI] [PubMed] [Google Scholar]
  4. Briggs L. M., Pecoraro V. L., McIntosh L. Copper-induced expression, cloning, and regulatory studies of the plastocyanin gene from the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol. 1990 Oct;15(4):633–642. doi: 10.1007/BF00017837. [DOI] [PubMed] [Google Scholar]
  5. Brock I. W., Hazell L., Michl D., Nielsen V. S., Møller B. L., Herrmann R. G., Klösgen R. B., Robinson C. Precursors of one integral and five lumenal thylakoid proteins are imported by isolated pea and barley thylakoids: optimisation of in vitro assays. Plant Mol Biol. 1993 Nov;23(4):717–725. doi: 10.1007/BF00021527. [DOI] [PubMed] [Google Scholar]
  6. Chitnis P. R., Purvis D., Nelson N. Molecular cloning and targeted mutagenesis of the gene psaF encoding subunit III of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem. 1991 Oct 25;266(30):20146–20151. [PubMed] [Google Scholar]
  7. 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]
  8. Creighton A. M., Hulford A., Mant A., Robinson D., Robinson C. A monomeric, tightly folded stromal intermediate on the delta pH-dependent thylakoidal protein transport pathway. J Biol Chem. 1995 Jan 27;270(4):1663–1669. doi: 10.1074/jbc.270.4.1663. [DOI] [PubMed] [Google Scholar]
  9. Haehnel W., Jansen T., Gause K., Klösgen R. B., Stahl B., Michl D., Huvermann B., Karas M., Herrmann R. G. Electron transfer from plastocyanin to photosystem I. EMBO J. 1994 Mar 1;13(5):1028–1038. doi: 10.1002/j.1460-2075.1994.tb06351.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Henry R., Kapazoglou A., McCaffery M., Cline K. Differences between lumen targeting domains of chloroplast transit peptides determine pathway specificity for thylakoid transport. J Biol Chem. 1994 Apr 8;269(14):10189–10192. [PubMed] [Google Scholar]
  11. 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]
  12. Inouye M., Halegoua S. Secretion and membrane localization of proteins in Escherichia coli. CRC Crit Rev Biochem. 1980;7(4):339–371. doi: 10.3109/10409238009105465. [DOI] [PubMed] [Google Scholar]
  13. Izard J. W., Kendall D. A. Signal peptides: exquisitely designed transport promoters. Mol Microbiol. 1994 Sep;13(5):765–773. doi: 10.1111/j.1365-2958.1994.tb00469.x. [DOI] [PubMed] [Google Scholar]
  14. James H. E., Bartling D., Musgrove J. E., Kirwin P. M., Herrmann R. G., Robinson C. Transport of proteins into chloroplasts. Import and maturation of precursors to the 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex. J Biol Chem. 1989 Nov 25;264(33):19573–19576. [PubMed] [Google Scholar]
  15. James H. E., Robinson C. Nucleotide sequence of cDNA encoding the precursor of the 23 kDa protein of the photosynthetic oxygen-evolving complex from wheat. Plant Mol Biol. 1991 Jul;17(1):179–182. doi: 10.1007/BF00036827. [DOI] [PubMed] [Google Scholar]
  16. Karnauchov I., Cai D., Schmidt I., Herrmann R. G., Klösgen R. B. The thylakoid translocation of subunit 3 of photosystem I, the psaF gene product, depends on a bipartite transit peptide and proceeds along an azide-sensitive pathway. J Biol Chem. 1994 Dec 30;269(52):32871–32878. [PubMed] [Google Scholar]
  17. 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]
  18. Knoetzel J., Simpson D. J. The primary structure of a cDNA for PsaN, encoding an extrinsic lumenal polypeptide of barley photosystem I. Plant Mol Biol. 1993 May;22(2):337–345. doi: 10.1007/BF00014940. [DOI] [PubMed] [Google Scholar]
  19. Knott T. G., Robinson C. The secA inhibitor, azide, reversibly blocks the translocation of a subset of proteins across the chloroplast thylakoid membrane. J Biol Chem. 1994 Mar 18;269(11):7843–7846. [PubMed] [Google Scholar]
  20. Kramer W., Drutsa V., Jansen H. W., Kramer B., Pflugfelder M., Fritz H. J. The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 1984 Dec 21;12(24):9441–9456. doi: 10.1093/nar/12.24.9441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kuwabara T., Reddy K. J., Sherman L. A. Nucleotide sequence of the gene from the cyanobacterium Anacystis nidulans R2 encoding the Mn-stabilizing protein involved in photosystem II water oxidation. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8230–8234. doi: 10.1073/pnas.84.23.8230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laudenbach D. E., Herbert S. K., McDowell C., Fork D. C., Grossman A. R., Straus N. A. Cytochrome c-553 is not required for photosynthetic activity in the cyanobacterium Synechococcus. Plant Cell. 1990 Sep;2(9):913–924. doi: 10.1105/tpc.2.9.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mant A., Nielsen V. S., Knott T. G., Møller B. L., Robinson C. Multiple mechanisms for the targeting of photosystem I subunits F, H, K, L, and N into and across the thylakoid membrane. J Biol Chem. 1994 Nov 4;269(44):27303–27309. [PubMed] [Google Scholar]
  24. Meadows J. W., Hulford A., Raines C. A., Robinson C. Nucleotide sequence of a cDNA clone encoding the precursor of the 33 kDa protein of the oxygen-evolving complex from wheat. Plant Mol Biol. 1991 Jun;16(6):1085–1087. doi: 10.1007/BF00016082. [DOI] [PubMed] [Google Scholar]
  25. Meadows J. W., Robinson C. The full precursor of the 33 kDa oxygen-evolving complex protein of wheat is exported by Escherichia coli and processed to the mature size. Plant Mol Biol. 1991 Dec;17(6):1241–1243. doi: 10.1007/BF00028739. [DOI] [PubMed] [Google Scholar]
  26. Michl D., Robinson C., Shackleton J. B., Herrmann R. G., Klösgen R. B. Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway. EMBO J. 1994 Mar 15;13(6):1310–1317. doi: 10.1002/j.1460-2075.1994.tb06383.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Nakai M., Goto A., Nohara T., Sugita D., Endo T. Identification of the SecA protein homolog in pea chloroplasts and its possible involvement in thylakoidal protein transport. J Biol Chem. 1994 Dec 16;269(50):31338–31341. [PubMed] [Google Scholar]
  30. Newman S. M., Eannetta N. T., Yu H., Prince J. P., de Vicente M. C., Tanksley S. D., Steffens J. C. Organisation of the tomato polyphenol oxidase gene family. Plant Mol Biol. 1993 Mar;21(6):1035–1051. doi: 10.1007/BF00023601. [DOI] [PubMed] [Google Scholar]
  31. Nielsen V. S., Mant A., Knoetzel J., Møller B. L., Robinson C. Import of barley photosystem I subunit N into the thylakoid lumen is mediated by a bipartite presequence lacking an intermediate processing site. Role of the delta pH in translocation across the thylakoid membrane. J Biol Chem. 1994 Feb 4;269(5):3762–3766. [PubMed] [Google Scholar]
  32. Philbrick J. B., Zilinskas B. A. Cloning, nucleotide sequence and mutational analysis of the gene encoding the Photosystem II manganese-stabilizing polypeptide of Synechocystis 6803. Mol Gen Genet. 1988 Jun;212(3):418–425. doi: 10.1007/BF00330845. [DOI] [PubMed] [Google Scholar]
  33. Robinson C., Cai D., Hulford A., Brock I. W., Michl D., Hazell L., Schmidt I., Herrmann R. G., Klösgen R. B. The presequence of a chimeric construct dictates which of two mechanisms are utilized for translocation across the thylakoid membrane: evidence for the existence of two distinct translocation systems. EMBO J. 1994 Jan 15;13(2):279–285. doi: 10.1002/j.1460-2075.1994.tb06260.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Robinson C., Klösgen R. B. Targeting of proteins into and across the thylakoid membrane--a multitude of mechanisms. Plant Mol Biol. 1994 Oct;26(1):15–24. doi: 10.1007/BF00039516. [DOI] [PubMed] [Google Scholar]
  35. Rother C., Jansen T., Tyagi A., Tittgen J., Herrmann R. G. Plastocyanin is encoded by an uninterrupted nuclear gene in spinach. Curr Genet. 1986;11(3):171–176. doi: 10.1007/BF00420603. [DOI] [PubMed] [Google Scholar]
  36. Sasaki S., Matsuyama S., Mizushima S. In vitro kinetic analysis of the role of the positive charge at the amino-terminal region of signal peptides in translocation of secretory protein across the cytoplasmic membrane in Escherichia coli. J Biol Chem. 1990 Mar 15;265(8):4358–4363. [PubMed] [Google Scholar]
  37. Seidler A., Michel H. Expression in Escherichia coli of the psbO gene encoding the 33 kd protein of the oxygen-evolving complex from spinach. EMBO J. 1990 Jun;9(6):1743–1748. doi: 10.1002/j.1460-2075.1990.tb08298.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shackleton J. B., Robinson C. Transport of proteins into chloroplasts. The thylakoidal processing peptidase is a signal-type peptidase with stringent substrate requirements at the -3 and -1 positions. J Biol Chem. 1991 Jul 5;266(19):12152–12156. [PubMed] [Google Scholar]
  39. Shestakov S. V., Anbudurai P. R., Stanbekova G. E., Gadzhiev A., Lind L. K., Pakrasi H. B. Molecular cloning and characterization of the ctpA gene encoding a carboxyl-terminal processing protease. Analysis of a spontaneous photosystem II-deficient mutant strain of the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem. 1994 Jul 29;269(30):19354–19359. [PubMed] [Google Scholar]
  40. Sommer A., Ne'eman E., Steffens J. C., Mayer A. M., Harel E. Import, targeting, and processing of a plant polyphenol oxidase. Plant Physiol. 1994 Aug;105(4):1301–1311. doi: 10.1104/pp.105.4.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Steppuhn J., Hermans J., Nechushtai R., Ljungberg U., Thümmler F., Lottspeich F., Herrmann R. G. Nucleotide sequence of cDNA clones encoding the entire precursor polypeptides for subunits IV and V of the photosystem I reaction center from spinach. FEBS Lett. 1988 Sep 12;237(1-2):218–224. doi: 10.1016/0014-5793(88)80205-9. [DOI] [PubMed] [Google Scholar]
  42. Theg S. M., Bauerle C., Olsen L. J., Selman B. R., Keegstra K. Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes. J Biol Chem. 1989 Apr 25;264(12):6730–6736. [PubMed] [Google Scholar]
  43. 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]
  44. Zhang L., Pakrasi H. B., Whitmarsh J. Photoautotrophic growth of the cyanobacterium Synechocystis sp. PCC 6803 in the absence of cytochrome c553 and plastocyanin. J Biol Chem. 1994 Feb 18;269(7):5036–5042. [PubMed] [Google Scholar]
  45. 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]

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