Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Nov 1;111(5):1825–1838. doi: 10.1083/jcb.111.5.1825

The chloroplast import receptor is an integral membrane protein of chloroplast envelope contact sites

PMCID: PMC2116326  PMID: 2172258

Abstract

A chloroplast import receptor from pea, previously identified by antiidiotypic antibodies was purified and its primary structure deduced from its cDNA sequence. The protein is a 36-kD integral membrane protein (p36) with eight potential transmembrane segments. Fab prepared from monospecific anti-p36 IgG inhibits the import of the ribulose-1,5- bisphosphate carboxylase small subunit precursor (pS) by interfering with pS binding at the chloroplast surface. Anti-p36 IgGs are able to immunoprecipitate a Triton X-100 soluble p36-pS complex, suggesting a direct interaction between p36 and pS. This immunoprecipitation was specific as it was abolished by a pS synthetic transit peptide, consistent with the transit sequence receptor function of p36. Immunoelectron microscopy localized p36 to regions of the outer chloroplast membrane that are in close contact with the inner chloroplast membrane. Comparison of the deduced sequence of pea p36 to that of other known proteins indicates a striking homology to a protein from spinach chloroplasts that was previously suggested to be the triose phosphate-3-phosphoglycerate-phosphate translocator (phosphate translocator) (Flugge, U. I., K. Fischer, A. Gross, W. Sebald, F. Lottspeich, and C. Eckerskorn. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:39-46). However, incubation of Triton X-100 solubilized chloroplast envelope material with hydroxylapatite indicated that p36 was quantitatively absorbed, whereas previous reports have shown that phosphate translocator activity does not bind to hydroxylapatite (Flugge, U. I., and H. W. Heldt. 1981. Biochim. Biophys. Acta. 638:296- 304. These data, in addition to the topology and import inhibition data presented in this report support the assignment of p36 as a receptor for chloroplast protein import, and argue against the assignment of the spinach homologue of this protein as the chloroplast phosphate translocator.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

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

  1. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bartel D., Lepke S., Layh-Schmitt G., Legrum B., Passow H. Anion transport in oocytes of Xenopus laevis induced by expression of mouse erythroid band 3 protein--encoding cRNA and of a cRNA derivative obtained by site-directed mutagenesis at the stilbene disulfonate binding site. EMBO J. 1989 Dec 1;8(12):3601–3609. doi: 10.1002/j.1460-2075.1989.tb08533.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chua N. H., Schmidt G. W. Transport of proteins into mitochondria and chloroplasts. J Cell Biol. 1979 Jun;81(3):461–483. doi: 10.1083/jcb.81.3.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Cornwell K. L., Keegstra K. Evidence that a Chloroplast Surface Protein Is Associated with a Specific Binding Site for the Precursor to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase. Plant Physiol. 1987 Nov;85(3):780–785. doi: 10.1104/pp.85.3.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Eisenberg D., Schwarz E., Komaromy M., Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. doi: 10.1016/0022-2836(84)90309-7. [DOI] [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Flügge U. I., Heldt H. W. Specific labelling of a protein involved in phosphate transport of chloroplasts by pyridoxal-5'-phosphate. FEBS Lett. 1977 Oct 1;82(1):29–33. doi: 10.1016/0014-5793(77)80878-8. [DOI] [PubMed] [Google Scholar]
  11. Friedman A. L., Keegstra K. Chloroplast protein import : quantitative analysis of precursor binding. Plant Physiol. 1989 Mar;89(3):993–999. doi: 10.1104/pp.89.3.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gratzer W. Membrane biology. The shadow and the substance. Nature. 1990 Feb 8;343(6258):514–514. doi: 10.1038/343514a0. [DOI] [PubMed] [Google Scholar]
  13. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  14. Hinz G., Flügge U. I. Phosphorylation of a 51-kDa envelope membrane polypeptide involved in protein translocation into chloroplasts. Eur J Biochem. 1988 Aug 15;175(3):649–659. doi: 10.1111/j.1432-1033.1988.tb14241.x. [DOI] [PubMed] [Google Scholar]
  15. Joyard J., Billecocq A., Bartlett S. G., Block M. A., Chua N. H., Douce R. Localization of polypeptides to the cytosolic side of the outer envelope membrane of spinach chloroplasts. J Biol Chem. 1983 Aug 25;258(16):10000–10006. [PubMed] [Google Scholar]
  16. 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]
  17. Keegstra K. Transport and routing of proteins into chloroplasts. Cell. 1989 Jan 27;56(2):247–253. doi: 10.1016/0092-8674(89)90898-2. [DOI] [PubMed] [Google Scholar]
  18. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  19. Lee C. C., Wu X. W., Gibbs R. A., Cook R. G., Muzny D. M., Caskey C. T. Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. Science. 1988 Mar 11;239(4845):1288–1291. doi: 10.1126/science.3344434. [DOI] [PubMed] [Google Scholar]
  20. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  21. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  22. Nikodem V., Fresco J. R. Protein fingerprinting by SDS-gel electrophoresis after partial fragmentation with CNBr. Anal Biochem. 1979 Sep 1;97(2):382–386. doi: 10.1016/0003-2697(79)90089-7. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Rumpho M. E., Edwards G. E. Characterization of 4,4'-Diisothiocyano-2,2'-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O(2) Evolution in Isolated Chloroplasts : Evidence for a Common Binding Site on the C(4) Phosphate Translocator for 3-Phosphoglycerate, Phosphoenolpyruvate, and Inorganic Phosphate. Plant Physiol. 1985 Jul;78(3):537–544. doi: 10.1104/pp.78.3.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rumpho M. E., Edwards G. E., Yousif A. E., Keegstra K. Specific Labeling of the Phosphate Translocator in C(3) and C(4) Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (1,2-Ditritio-1,2-[2,2' -Disulfo-4,4' -Diisothiocyano] Diphenylethane). Plant Physiol. 1988 Apr;86(4):1193–1198. doi: 10.1104/pp.86.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Schleyer M., Neupert W. Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes. Cell. 1985 Nov;43(1):339–350. doi: 10.1016/0092-8674(85)90039-x. [DOI] [PubMed] [Google Scholar]
  30. Schmidt G. W., Mishkind M. L. The transport of proteins into chloroplasts. Annu Rev Biochem. 1986;55:879–912. doi: 10.1146/annurev.bi.55.070186.004311. [DOI] [PubMed] [Google Scholar]
  31. Schwaiger M., Herzog V., Neupert W. Characterization of translocation contact sites involved in the import of mitochondrial proteins. J Cell Biol. 1987 Jul;105(1):235–246. doi: 10.1083/jcb.105.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Wood W. I. Gene cloning based on long oligonucleotide probes. Methods Enzymol. 1987;152:443–447. doi: 10.1016/0076-6879(87)52051-1. [DOI] [PubMed] [Google Scholar]
  34. van Berkel J., Steup M., Völker W., Robenek H., Flügge U. I. Polypeptides of the chloroplast envelope membranes as visualized by immunochemical techniques. J Histochem Cytochem. 1986 May;34(5):577–583. doi: 10.1177/34.5.3517143. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES