RanBP1 stabilizes the interaction of Ran with p97 nuclear protein import

Three factors have been identified that reconstitute nuclear protein import in a permeabilized cell assay: the NLS receptor, p97, and Ran/TC4. Ran/TC4, in turn, interacts with a number of proteins that are involved in the regulation of GTP hydrolysis or are components of the nuclear pore. Two Ran-binding proteins, RanBP1 and RanBP2, form discrete complexes with p97 as demonstrated by immunoadsorption from HeLa cell extracts fractionated by gel filtration chromatography. A > 400-kD complex contains p97, Ran, and RanBP2. Another complex of 150- 300 kD was comprised of p97, Ran, and RanBP1. This second trimeric complex could be reconstituted from recombinant proteins. In solution binding assays, Ran-GTP bound p97 with high affinity, but the binding of Ran-GDP to p97 was undetectable. The addition of RanBP1 with Ran-GDP or Ran-GTP increased the affinity of both forms of Ran for p97 to the same level. Binding of Ran-GTP to p97 dissociated p97 from immobilized NLS receptor while the Ran-GDP/RanBP1/p97 complex did not dissociate from the receptor. In a digitonin-permeabilized cell docking assay, RanBP1 stabilizes the receptor complex against temperature-dependent release from the pore. When added to an import assay with recombinant NLS receptor, p97 and Ran-GDP, RanBP1 significantly stimulates transport. These results suggest that RanBP1 promotes both the docking and translocation steps in nuclear protein import by stabilizing the interaction of Ran-GDP with p97.

[1]  G. Blobel,et al.  Role of the Nuclear Transport Factor p10 in Nuclear Import , 1996, Science.

[2]  A. Lamond,et al.  The conserved amino‐terminal domain of hSRP1 alpha is essential for nuclear protein import. , 1996, The EMBO journal.

[3]  E. Hartmann,et al.  A 41 amino acid motif in importin‐alpha confers binding to importin‐beta and hence transit into the nucleus. , 1996, The EMBO journal.

[4]  G. Blobel,et al.  The Nuclear Transport Factor Karyopherin Binds Stoichiometrically to Ran-GTP and Inhibits the Ran GTPase Activating Protein (*) , 1996, The Journal of Biological Chemistry.

[5]  I. Macara,et al.  Ran Binding Domains Promote the Interaction of Ran with p97/β-Karyopherin, Linking the Docking and Translocation Steps of Nuclear Import (*) , 1996, The Journal of Biological Chemistry.

[6]  J. L. Watkins,et al.  The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor , 1995, The Journal of cell biology.

[7]  G. Blobel,et al.  Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins , 1995, Cell.

[8]  F. Melchior,et al.  GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import , 1995, The Journal of cell biology.

[9]  P. Silver,et al.  Mutants in a yeast Ran binding protein are defective in nuclear transport. , 1995, The EMBO journal.

[10]  G. Blobel,et al.  Nuclear Import of Influenza Virus RNA Can Be Mediated by Viral Nucleoprotein and Transport Factors Required for Protein Import (*) , 1995, The Journal of Biological Chemistry.

[11]  P. Silver,et al.  Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import , 1995, The Journal of cell biology.

[12]  E. Hartmann,et al.  Distinct functions for the two importin subunits in nuclear protein import , 1995, Nature.

[13]  B. Séraphin,et al.  A family of Ran binding proteins that includes nucleoporins. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  G. Blobel,et al.  Identification of a Yeast Karyopherin Heterodimer That Targets Import Substrate to Mammalian Nuclear Pore Complexes (*) , 1995, The Journal of Biological Chemistry.

[15]  Tatsuya Seki,et al.  A giant nucleopore protein that binds Ran/TC4 , 1995, Nature.

[16]  S. Adam,et al.  Sequence and characterization of cytoplasmic nuclear protein import factor p97 , 1995, The Journal of cell biology.

[17]  I. Macara,et al.  The C Terminus of the Nuclear RAN/TC4 GTPase Stabilizes the GDP-bound State and Mediates Interactions with RCC1, RAN-GAP, and HTF9A/RANBP1 (*) , 1995, The Journal of Biological Chemistry.

[18]  G. Blobel,et al.  Nup358, a Cytoplasmically Exposed Nucleoporin with Peptide Repeats, Ran-GTP Binding Sites, Zinc Fingers, a Cyclophilin A Homologous Domain, and a Leucine-rich Region (*) , 1995, The Journal of Biological Chemistry.

[19]  F. Bischoff,et al.  RNA1 Encodes a GTPase-activating Protein Specific for Gsp1p, the Ran/TC4 Homologue of Saccharomyces cerevisiae(*) , 1995, The Journal of Biological Chemistry.

[20]  A. Lamond,et al.  Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences. , 1995, Science.

[21]  M. Dasso,et al.  The RCC1 Protein Interacts with Ran, RanBP1, hsc70, and a 340-kDa Protein in Xenopus Extracts (*) , 1995, The Journal of Biological Chemistry.

[22]  B. Paschal,et al.  Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62 , 1995, The Journal of cell biology.

[23]  E. Hartmann,et al.  A Ran-binding motif in nuclear pore proteins. , 1995, Trends in cell biology.

[24]  G. Blobel,et al.  The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex , 1995, Cell.

[25]  N. Imamoto,et al.  A Karyophilic Protein Forms a Stable Complex with Cytoplasmic Components Prior to Nuclear Pore Binding (*) , 1995, The Journal of Biological Chemistry.

[26]  I. Macara,et al.  The Ran/TC4 GTPase-binding domain: identification by expression cloning and characterization of a conserved sequence motif. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[27]  P. D’Eustachio,et al.  Separate domains of the Ran GTPase interact with different factors to regulate nuclear protein import and RNA processing , 1995, Molecular and cellular biology.

[28]  R. Kraft,et al.  Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope , 1995, Current Biology.

[29]  G. Blobel,et al.  Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[30]  G. Blobel,et al.  Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[31]  F. Bischoff,et al.  Co‐activation of RanGTPase and inhibition of GTP dissociation by Ran‐GTP binding protein RanBP1. , 1995, The EMBO journal.

[32]  P. Palese,et al.  NPI-1, the human homolog of SRP-1, interacts with influenza virus nucleoprotein. , 1995, Virology.

[33]  M. Powers,et al.  Cytosolic factors in nuclear transport: What's importin? , 1994, Cell.

[34]  E. Hartmann,et al.  Isolation of a protein that is essential for the first step of nuclear protein import , 1994, Cell.

[35]  G. Blobel,et al.  Purification of a Ran-interacting protein that is required for protein import into the nucleus. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[36]  S. Adam,et al.  Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope , 1994, The Journal of cell biology.

[37]  I. Macara,et al.  A family of proteins that stabilize the Ran/TC4 GTPase in its GTP-bound conformation. , 1994, The Journal of biological chemistry.

[38]  F. Bischoff,et al.  RanGAP1 induces GTPase activity of nuclear Ras-related Ran. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[39]  P. D’Eustachio,et al.  Characterization of proteins that interact with the cell-cycle regulatory protein Ran/TC4 , 1993, Nature.

[40]  F. Melchior,et al.  Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor [published erratum appears in J Cell Biol 1994 Jan;124(1-2):217] , 1993, The Journal of cell biology.

[41]  G. Blobel,et al.  The GTP-binding protein Ran/TC4 is required for protein import into the nucleus , 1993, Nature.

[42]  D. Newmeyer.  The nuclear pore complex and nucleocytoplasmic transport. , 1993, Current opinion in cell biology.

[43]  F. Bischoff,et al.  Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[44]  F. Bischoff,et al.  Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1 , 1991, Nature.

[45]  S. Adam,et al.  Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import , 1991, Cell.

[46]  Frank McCormick,et al.  The GTPase superfamily: a conserved switch for diverse cell functions , 1990, Nature.

[47]  S. Adam,et al.  Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors , 1990, The Journal of cell biology.

[48]  C. Feldherr,et al.  Movement of a karyophilic protein through the nuclear pores of oocytes , 1984, The Journal of cell biology.

[49]  G. Dreyfuss,et al.  Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription , 1984, Molecular and cellular biology.

[50]  F. Melchior,et al.  Analysis of Ran/TC4 function in nuclear protein import. , 1995, Methods in enzymology.

[51]  F. Melchior,et al.  Mechanisms of nuclear protein import. , 1995, Current opinion in cell biology.

[52]  G. Blobel,et al.  Soluble factors required for nuclear protein import. , 1995, Cold Spring Harbor symposia on quantitative biology.

[53]  R. Schneiter,et al.  The nuclear GTPase cycle: promoting peripheralization? , 1995, Trends in cell biology.

[54]  D. Koepp,et al.  Rnalp, a Ran/tc4 Gtpase Activating Protein, Is Required for Nuclear Import , 1995 .

[55]  R. Schneppenheim,et al.  Luminography – a new, highly sensitive visualization method for electrophoresis , 1991, Electrophoresis.

[56]  R. Laskey,et al.  Nuclear targeting sequences--a consensus? , 1991, Trends in biochemical sciences.

[57]  Deutsches Krebsforschungszentrum,et al.  Nucleocytoplasmic Transport , 1986, Springer Berlin Heidelberg.

[58]  E. Hartmann,et al.  A 41 amino acid motif in importin-a confers binding to importin-, and hence transit into the nucleus , 2022 .