Skip to main content
The Plant Cell logoLink to The Plant Cell
. 1995 Sep;7(9):1421–1432. doi: 10.1105/tpc.7.9.1421

The N-terminal hydrophobic region of the mature phosphate translocator is sufficient for targeting to the chloroplast inner envelope membrane.

J S Knight 1, J C Gray 1
PMCID: PMC160965  PMID: 8589626

Abstract

To locate the sequence required for directing the phosphate translocator to the chloroplast inner envelope membrane, a series of chimeric proteins constituting parts of the phosphate translocator and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, which is normally located in the stroma, has been produced. Reciprocal exchanges of the presequences and mature sequences of the phosphate translocator and the small subunit indicated that the phosphate translocator presequence contains stromal targeting information and that the mature protein is responsible for inner envelope membrane targeting. Chimeric proteins containing the N-terminal 46 amino acid residues of the phosphate translocator were directed to the inner envelope membrane. Subdivision of this region into its composite hydrophilic and hydrophobic regions showed that the hydrophobic region alone, which consists of amino acid residues 24 to 45, was able to direct the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to the inner envelope membrane.

Full Text

The Full Text of this article is available as a PDF (3.3 MB).

Selected References

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

  1. Anderson S., Smith S. M. Synthesis of the small subunit of ribulose-bisphosphate carboxylase from genes cloned into plasmids containing the SP6 promoter. Biochem J. 1986 Dec 15;240(3):709–715. doi: 10.1042/bj2400709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrews J., Ohlrogge J. B., Keegstra K. Final step of phosphatidic Acid synthesis in pea chloroplasts occurs in the inner envelope membrane. Plant Physiol. 1985 Jul;78(3):459–465. doi: 10.1104/pp.78.3.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barnes S. A., Knight J. S., Gray J. C. Alteration of the Amount of the Chloroplast Phosphate Translocator in Transgenic Tobacco Affects the Distribution of Assimilate between Starch and Sugar. Plant Physiol. 1994 Nov;106(3):1123–1129. doi: 10.1104/pp.106.3.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beasley E. M., Müller S., Schatz G. The signal that sorts yeast cytochrome b2 to the mitochondrial intermembrane space contains three distinct functional regions. EMBO J. 1993 Jun;12(6):2303–2311. doi: 10.1002/j.1460-2075.1993.tb05884.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beltzer J. P., Fiedler K., Fuhrer C., Geffen I., Handschin C., Wessels H. P., Spiess M. Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence. J Biol Chem. 1991 Jan 15;266(2):973–978. [PubMed] [Google Scholar]
  6. Cheung A. Y., Bogorad L., Van Montagu M., Schell J. Relocating a gene for herbicide tolerance: A chloroplast gene is converted into a nuclear gene. Proc Natl Acad Sci U S A. 1988 Jan;85(2):391–395. doi: 10.1073/pnas.85.2.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Douce R., Holtz R. B., Benson A. A. Isolation and properties of the envelope of spinach chloroplasts. J Biol Chem. 1973 Oct 25;248(20):7215–7222. [PubMed] [Google Scholar]
  10. 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]
  11. Dreses-Werringloer U., Fischer K., Wachter E., Link T. A., Flügge U. I. cDNA sequence and deduced amino acid sequence of the precursor of the 37-kDa inner envelope membrane polypeptide from spinach chloroplasts. Its transit peptide contains an amphiphilic alpha-helix as the only detectable structural element. Eur J Biochem. 1991 Jan 30;195(2):361–368. doi: 10.1111/j.1432-1033.1991.tb15714.x. [DOI] [PubMed] [Google Scholar]
  12. Fischer K., Weber A., Arbinger B., Brink S., Eckerskorn C., Flügge U. I. The 24 kDa outer envelope membrane protein from spinach chloroplasts: molecular cloning, in vivo expression and import pathway of a protein with unusual properties. Plant Mol Biol. 1994 May;25(2):167–177. doi: 10.1007/BF00023235. [DOI] [PubMed] [Google Scholar]
  13. Fliege R., Flügge U. I., Werdan K., Heldt H. W. Specific transport of inorganic phosphate, 3-phosphoglycerate and triosephosphates across the inner membrane of the envelope in spinach chloroplasts. Biochim Biophys Acta. 1978 May 10;502(2):232–247. doi: 10.1016/0005-2728(78)90045-2. [DOI] [PubMed] [Google Scholar]
  14. Flügge U. I., Fischer K., Gross A., Sebald W., Lottspeich F., Eckerskorn C. The triose phosphate-3-phosphoglycerate-phosphate translocator from spinach chloroplasts: nucleotide sequence of a full-length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts. EMBO J. 1989 Jan;8(1):39–46. doi: 10.1002/j.1460-2075.1989.tb03346.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gavel Y., Steppuhn J., Herrmann R., von Heijne G. The 'positive-inside rule' applies to thylakoid membrane proteins. FEBS Lett. 1991 Apr 22;282(1):41–46. doi: 10.1016/0014-5793(91)80440-e. [DOI] [PubMed] [Google Scholar]
  16. Gavel Y., von Heijne G. The distribution of charged amino acids in mitochondrial inner-membrane proteins suggests different modes of membrane integration for nuclearly and mitochondrially encoded proteins. Eur J Biochem. 1992 May 1;205(3):1207–1215. doi: 10.1111/j.1432-1033.1992.tb16892.x. [DOI] [PubMed] [Google Scholar]
  17. Gearing D. P., Nagley P. Yeast mitochondrial ATPase subunit 8, normally a mitochondrial gene product, expressed in vitro and imported back into the organelle. EMBO J. 1986 Dec 20;5(13):3651–3655. doi: 10.1002/j.1460-2075.1986.tb04695.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Glick B. S., Beasley E. M., Schatz G. Protein sorting in mitochondria. Trends Biochem Sci. 1992 Nov;17(11):453–459. doi: 10.1016/0968-0004(92)90487-t. [DOI] [PubMed] [Google Scholar]
  19. Heijne G. The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. EMBO J. 1986 Nov;5(11):3021–3027. doi: 10.1002/j.1460-2075.1986.tb04601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hirsch S., Muckel E., Heemeyer F., von Heijne G., Soll J. A receptor component of the chloroplast protein translocation machinery. Science. 1994 Dec 23;266(5193):1989–1992. doi: 10.1126/science.7801125. [DOI] [PubMed] [Google Scholar]
  21. Jensen R. E., Schmidt S., Mark R. J. Mutations in a 19-amino-acid hydrophobic region of the yeast cytochrome c1 presequence prevent sorting to the mitochondrial intermembrane space. Mol Cell Biol. 1992 Oct;12(10):4677–4686. doi: 10.1128/mcb.12.10.4677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Joyard J., Grossman A., Bartlett S. G., Douce R., Chua N. H. Characterization of envelope membrane polypeptides from spinach chloroplasts. J Biol Chem. 1982 Jan 25;257(2):1095–1101. [PubMed] [Google Scholar]
  23. Knight J. S., Gray J. C. Expression of genes encoding the tobacco chloroplast phosphate translocator is not light-regulated and is repressed by sucrose. Mol Gen Genet. 1994 Mar;242(5):586–594. doi: 10.1007/BF00285282. [DOI] [PubMed] [Google Scholar]
  24. Ko K., Bornemisza O., Kourtz L., Ko Z. W., Plaxton W. C., Cashmore A. R. Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope. J Biol Chem. 1992 Feb 15;267(5):2986–2993. [PubMed] [Google Scholar]
  25. 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]
  26. Kuroiwa T., Sakaguchi M., Mihara K., Omura T. Structural requirements for interruption of protein translocation across rough endoplasmic reticulum membrane. J Biochem. 1990 Nov;108(5):829–834. doi: 10.1093/oxfordjournals.jbchem.a123288. [DOI] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Li H. M., Moore T., Keegstra K. Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts. Plant Cell. 1991 Jul;3(7):709–717. doi: 10.1105/tpc.3.7.709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Madueno F., Napier J. A., Gray J. C. Newly Imported Rieske Iron-Sulfur Protein Associates with Both Cpn60 and Hsp70 in the Chloroplast Stroma. Plant Cell. 1993 Dec;5(12):1865–1876. doi: 10.1105/tpc.5.12.1865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Madueño F., Napier J. A., Cejudo F. J., Gray J. C. Import and processing of the precursor of the Rieske FeS protein of tobacco chloroplasts. Plant Mol Biol. 1992 Oct;20(2):289–299. doi: 10.1007/BF00014496. [DOI] [PubMed] [Google Scholar]
  31. Murakami S., Strotmann H. Adenylate kinase bound to the envelope membranes of spinach chloroplasts. Arch Biochem Biophys. 1978 Jan 15;185(1):30–38. doi: 10.1016/0003-9861(78)90140-6. [DOI] [PubMed] [Google Scholar]
  32. Oblong J. E., Lamppa G. K. Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast. EMBO J. 1992 Dec;11(12):4401–4409. doi: 10.1002/j.1460-2075.1992.tb05540.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Perry S. E., Keegstra K. Envelope membrane proteins that interact with chloroplastic precursor proteins. Plant Cell. 1994 Jan;6(1):93–105. doi: 10.1105/tpc.6.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  35. Pfanner N., Hoeben P., Tropschug M., Neupert W. The carboxyl-terminal two-thirds of the ADP/ATP carrier polypeptide contains sufficient information to direct translocation into mitochondria. J Biol Chem. 1987 Nov 5;262(31):14851–14854. [PubMed] [Google Scholar]
  36. Salomon M., Fischer K., Flügge U. I., Soll J. Sequence analysis and protein import studies of an outer chloroplast envelope polypeptide. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5778–5782. doi: 10.1073/pnas.87.15.5778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schnell D. J., Kessler F., Blobel G. Isolation of components of the chloroplast protein import machinery. Science. 1994 Nov 11;266(5187):1007–1012. doi: 10.1126/science.7973649. [DOI] [PubMed] [Google Scholar]
  38. Schulz B., Frommer W. B., Flügge U. I., Hummel S., Fischer K., Willmitzer L. Expression of the triose phosphate translocator gene from potato is light dependent and restricted to green tissues. Mol Gen Genet. 1993 Apr;238(3):357–361. doi: 10.1007/BF00291994. [DOI] [PubMed] [Google Scholar]
  39. Steck T. L., Yu J. Selective solubilization of proteins from red blood cell membranes by protein perturbants. J Supramol Struct. 1973;1(3):220–232. doi: 10.1002/jss.400010307. [DOI] [PubMed] [Google Scholar]
  40. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wu C., Seibert F. S., Ko K. Identification of chloroplast envelope proteins in close physical proximity to a partially translocated chimeric precursor protein. J Biol Chem. 1994 Dec 23;269(51):32264–32271. [PubMed] [Google Scholar]
  42. von Heijne G., Gavel Y. Topogenic signals in integral membrane proteins. Eur J Biochem. 1988 Jul 1;174(4):671–678. doi: 10.1111/j.1432-1033.1988.tb14150.x. [DOI] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES