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PMID: 8776900 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S.

Nuclear import in permeabilized protoplasts from higher plants has unique features.

The Plant cell ·Vol. 8 ·No. 8 ·1996-08-00 ·Pages 1337-52

Hicks GR, Smith HM, Lobreaux S, Raikhel NV

Abstract

The import of proteins into the nucleus is a poorly understood process that is thought to require soluble cytosolic factors in vertebrates and yeast. To test this model in plants and to identify components of the import apparatus, we developed a direct in vitro nuclear import assay by using tobacco protoplasts that were permeabilized without detergents such as digitonin or Triton X-100. Substrates were imported specifically by a mechanism that required only guanine nucleotides. Moreover, in vitro import did not require exogenous cytosol. To investigate this novel finding, we isolated a full-length cDNA encoding an Arabidopsis homolog of vertebrate and yeast nuclear localization signal receptors and produced an affinity-purified antibody. The plant receptor was tightly associated with cellular components in permeabilized protoplasts, even in the presence of 0.1% Triton X-100, indicating that this factor and probably others were retained to an extent sufficient to support import. The lectin wheat germ agglutinin bound to the nucleus; however, it did not block translocation in our system, indicating that direct interaction with polysaccharide modifications at the nuclear pore complex was probably not essential for import in plants. Other features of in vitro import included reduced but significant import at low temperature.

MeSH Terms
Amino Acid Sequence Biological Transport, Active Cell Nucleus/metabolism Cytosol/metabolism Guanine Nucleotides/metabolism Karyopherins Molecular Sequence Data Nuclear Localization Signals Nuclear Proteins/genetics,metabolism Permeability Plant Proteins/genetics,metabolism Plants/genetics,metabolism Plants, Toxic Protoplasts/metabolism Sequence Homology, Amino Acid Tobacco
Chemicals
Guanine Nucleotides Karyopherins Nuclear Localization Signals Nuclear Proteins Plant Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Hicks G R
Michigan State University-Department of Energy, East Lansing 48824-1312, USA.
Smith H M
Lobreaux S
Raikhel N V
References (64)
64 references, click to expand
  1. The GTP-bound form of the yeast Ran/TC4 homologue blocks nuclear protein import and appearance of poly(A)+ RNA in the cytoplasm.
    Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):225-9 PMID: 7816822
  2. The basic domain in the bZIP regulatory protein Opaque2 serves two independent functions: DNA binding and nuclear localization.
    Plant J. 1994 Feb;5(2):207-14 PMID: 8148877
  3. A nuclear localization domain in the hnRNP A1 protein.
    J Cell Biol. 1995 May;129(3):551-60 PMID: 7730395
  4. Plant nuclear pore complex proteins are modified by novel oligosaccharides with terminal N-acetylglucosamine.
    Plant Cell. 1995 Sep;7(9):1459-71 PMID: 8589629
  5. A family of Ran binding proteins that includes nucleoporins.
    Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7525-9 PMID: 7638224
  6. Nup358, a cytoplasmically exposed nucleoporin with peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine-rich region.
    J Biol Chem. 1995 Jun 9;270(23):14209-13 PMID: 7775481
  7. The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor.
    J Cell Biol. 1995 Dec;131(6 Pt 2):1699-713 PMID: 8557738
  8. Sequence and characterization of cytoplasmic nuclear protein import factor p97.
    J Cell Biol. 1995 Jul;130(2):265-74 PMID: 7615630
  9. In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins.
    J Cell Biol. 1986 Dec;103(6 Pt 1):2091-102 PMID: 3097026
  10. The nuclear pore-targeting complex binds to nuclear pores after association with a karyophile.
    FEBS Lett. 1995 Jul 24;368(3):415-9 PMID: 7635189
  11. Nucleocytoplasmic transport.
    Biochem J. 1994 Jun 15;300 ( Pt 3):609-18 PMID: 8010940
  12. Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins.
    Cell. 1995 Dec 1;83(5):683-92 PMID: 8521485
  13. Apparent Inhibition of Chloroplast Protein Import by Cold Temperatures Is Due to Energetic Considerations Not Membrane Fluidity.
    Plant Cell. 1994 Mar;6(3):427-437 PMID: 12244243
  14. Facilitated nuclear transport of calmodulin in tissue culture cells.
    J Cell Biol. 1994 Dec;127(6 Pt 1):1527-36 PMID: 7798309
  15. Nuclear targeting sequences--a consensus?
    Trends Biochem Sci. 1991 Dec;16(12):478-81 PMID: 1664152
  16. Tissue-specific differences of the mitochondrial protein import machinery: in vitro import, processing and degradation of the pre-F1 beta subunit of the ATP synthase in spinach leaf and root mitochondria.
    Plant Mol Biol. 1994 Oct;26(2):571-9 PMID: 7948913
  17. Light-regulated modification and nuclear translocation of cytosolic G-box binding factors in parsley.
    Plant Cell. 1994 Apr;6(4):545-59 PMID: 8205004
  18. GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import.
    J Cell Biol. 1995 Nov;131(3):571-81 PMID: 7593180
  19. A giant nucleopore protein that binds Ran/TC4.
    Nature. 1995 Jul 13;376(6536):184-8 PMID: 7603572
  20. A small nuclear GTP-binding protein from tomato suppresses a Schizosaccharomyces pombe cell-cycle mutant.
    Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5863-7 PMID: 8016079
  21. Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor.
    J Cell Biol. 1993 Dec;123(6 Pt 2):1649-59 PMID: 8276887
  22. Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins.
    Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1769-73 PMID: 7878057
  23. Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells.
    Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2392-7 PMID: 8637884
  24. Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences.
    Science. 1995 May 19;268(5213):1049-53 PMID: 7754385
  25. RAG-1 interacts with the repeated amino acid motif of the human homologue of the yeast protein SRP1.
    Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7633-7 PMID: 8052633
  26. The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex.
    Cell. 1995 Apr 21;81(2):215-22 PMID: 7736573
  27. Pendulin, a Drosophila protein with cell cycle-dependent nuclear localization, is required for normal cell proliferation.
    J Cell Biol. 1995 Jun;129(6):1491-507 PMID: 7790350
  28. Functional nuclear pores reconstituted with beta 1-4 galactose-modified O-linked N-acetylglucosamine glycoproteins.
    J Biol Chem. 1994 Mar 25;269(12):9289-97 PMID: 8132666
  29. Purification of a Ran-interacting protein that is required for protein import into the nucleus.
    Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10212-6 PMID: 7937864
  30. Protein import into the nucleus: an integrated view.
    Annu Rev Cell Dev Biol. 1995;11:155-88 PMID: 8689555
  31. The yeast nuclear import receptor is required for mitosis.
    Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7647-51 PMID: 7644471
  32. One of three nuclear localization signals of maize Activator (Ac) transposase overlaps the DNA-binding domain.
    Plant J. 1995 Mar;7(3):441-51 PMID: 7757116
  33. Identification of specific binding proteins for a nuclear location sequence.
    Nature. 1989 Jan 19;337(6204):276-9 PMID: 2911368
  34. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors.
    J Cell Biol. 1990 Sep;111(3):807-16 PMID: 2391365
  35. Specific binding of nuclear localization sequences to plant nuclei.
    Plant Cell. 1993 Aug;5(8):983-94 PMID: 8400874
  36. Monoclonal anti-histone autoantibodies derived from murine models of lupus.
    J Immunol. 1984 Nov;133(5):2554-9 PMID: 6332855
  37. Reconstitution of nuclear protein transport with semi-intact yeast cells.
    J Cell Biol. 1993 Nov;123(4):785-98 PMID: 8227140
  38. Three classes of nuclear import signals bind to plant nuclei.
    Plant Physiol. 1995 Apr;107(4):1055-8 PMID: 7770516
  39. Mechanisms of nuclear protein import.
    Curr Opin Cell Biol. 1995 Jun;7(3):310-8 PMID: 7662359
  40. Distinct functions for the two importin subunits in nuclear protein import.
    Nature. 1995 Sep 21;377(6546):246-8 PMID: 7675110
  41. Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope.
    Curr Biol. 1995 Apr 1;5(4):383-92 PMID: 7627554
  42. Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae.
    Mol Cell Biol. 1992 Dec;12(12):5640-51 PMID: 1448093
  43. PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.
    J Cell Biol. 1996 Feb;132(3):359-70 PMID: 8636214
  44. Nucleocytoplasmic transport.
    Science. 1996 Mar 15;271(5255):1513-8 PMID: 8599106
  45. Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import.
    Cell. 1991 Sep 6;66(5):837-47 PMID: 1653647
  46. The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate.
    Mol Cell Biol. 1992 May;12(5):2186-92 PMID: 1569948
  47. A 41 amino acid motif in importin-alpha confers binding to importin-beta and hence transit into the nucleus.
    EMBO J. 1996 Apr 15;15(8):1810-7 PMID: 8617226
  48. Phenotype of the fission yeast cell cycle regulatory mutant pim1-46 is suppressed by a tobacco cDNA encoding a small, Ran-like GTP-binding protein.
    Plant J. 1994 Oct;6(4):555-65 PMID: 7987414
  49. Unidirectional digestion with exonuclease III in DNA sequence analysis.
    Methods Enzymol. 1987;155:156-65 PMID: 3323819
  50. The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import.
    EMBO J. 1996 Apr 15;15(8):1818-25 PMID: 8617227
  51. Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope.
    J Cell Biol. 1994 May;125(3):547-55 PMID: 8175880
  52. Identification of a yeast karyopherin heterodimer that targets import substrate to mammalian nuclear pore complexes.
    J Biol Chem. 1995 Jul 14;270(28):16499-502 PMID: 7622450
  53. Structure and function of the nuclear pore complex.
    Annu Rev Cell Biol. 1992;8:495-527 PMID: 1282353
  54. The importance of importin.
    Trends Cell Biol. 1995 May;5(5):189-91 PMID: 14731446
  55. Role of the nuclear transport factor p10 in nuclear import.
    Science. 1996 Apr 5;272(5258):120-2 PMID: 8600522
  56. Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62.
    J Cell Biol. 1995 May;129(4):925-37 PMID: 7744965
  57. Putative nuclear localization signals (NLS) in protein transcription factors.
    J Cell Biochem. 1994 May;55(1):32-58 PMID: 8083298
  58. A repeating amino acid motif shared by proteins with diverse cellular roles.
    Cell. 1994 Mar 11;76(5):789-91 PMID: 7907279
  59. Artifactual detection of ADP-dependent sucrose synthase in crude plant extracts.
    FEBS Lett. 1992 Sep 14;309(3):283-7 PMID: 1387619
  60. The GTP-binding protein Ran/TC4 is required for protein import into the nucleus.
    Nature. 1993 Oct 14;365(6447):661-3 PMID: 8413630
  61. Nuclear localization signals (NLS).
    Crit Rev Eukaryot Gene Expr. 1993;3(3):193-227 PMID: 8241603
  62. Isolation of a protein that is essential for the first step of nuclear protein import.
    Cell. 1994 Dec 2;79(5):767-78 PMID: 8001116
  63. Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.
    J Cell Biol. 1987 Feb;104(2):189-200 PMID: 3805121
  64. The regulation of protein transport to the nucleus by phosphorylation.
    Biochem J. 1995 Nov 1;311 ( Pt 3):705-16 PMID: 7487922
Article Info
Journal
The Plant cell
Abbr.
Plant Cell
ISSN
1040-4651
DOI
10.1105/tpc.8.8.1337
Published
1996-08-00
Pages
1337-52
Language
English
Region
England
NLM ID
9208688
PMCID
PMC161251
Subset
IM
Databases
GENBANK
AF077528
External Links
PubMed source DOI: 10.1105/tpc.8.8.1337 PMC Full Text View journal details
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