Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin-beta but interacts differently with RanBP1

We report the identification and characterization of a novel 124-kDa Ran binding protein, RanBP5. This protein is related to importin-beta, the key mediator of nuclear localization signal (NLS)-dependent nuclear transport. RanBP5 was identified by two independent methods: it was isolated from HeLa cells by using its interaction with RanGTP in an overlay assay to monitor enrichment, and it was also found by the yeast two-hybrid selection method with RanBP1 as bait. RanBP5 binds to RanBP1 as part of a trimeric RanBP1-Ran-RanBP5 complex. Like importin-beta, RanBP5 strongly binds the GTP-bound form of Ran, stabilizing it against both intrinsic and RanGAP1-induced GTP hydrolysis and also against nucleotide exchange. The GAP resistance of the RanBP5-RanGTP complex can be relieved by RanBP1, which might reflect an in vivo role for RanBP1. RanBP5 is a predominantly cytoplasmic protein that can bind to nuclear pore complexes. We propose that RanBP5 is a mediator of a nucleocytoplasmic transport pathway that is distinct from the importin-alpha-dependent import of proteins with a classical NLS.

[1]  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.

[2]  C. Klebe,et al.  Functional expression in Escherichia coli of the mitotic regulator proteins p24ran and p45rcc1 and fluorescence measurements of their interaction. , 1993, Biochemistry.

[3]  R. Goody,et al.  The kinetic mechanism of Ran--nucleotide exchange catalyzed by RCC1. , 1995, Biochemistry.

[4]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[5]  S. Elledge,et al.  The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. , 1993, Genes & development.

[6]  M. Dasso,et al.  RanBP2 associates with Ubc9p and a modified form of RanGAP1. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[7]  G. Blobel,et al.  A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex , 1996, The Journal of cell biology.

[8]  M. Fornerod,et al.  The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88 , 1997, The EMBO journal.

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

[10]  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.

[11]  D. Dignard,et al.  Screening and identification of a gene, PSE-1, that affects protein secretion in Saccharomyces cerevisiae. , 1992, Journal of cell science.

[12]  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.

[13]  P. D’Eustachio,et al.  The small nuclear GTPase Ran: How much does it run? , 1996, BioEssays : news and reviews in molecular, cellular and developmental biology.

[14]  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.

[15]  G. Blobel,et al.  A Distinct Nuclear Import Pathway Used by Ribosomal Proteins , 1997, Cell.

[16]  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.

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

[18]  S. Elledge,et al.  The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases , 1993, Cell.

[19]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[20]  M. Kozak.  An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. , 1987, Nucleic acids research.

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

[22]  I. Macara,et al.  Ran-binding Protein 1 (RanBP1) Forms a Ternary Complex with Ran and Karyopherin β and Reduces Ran GTPase-activating Protein (RanGAP) Inhibition by Karyopherin β* , 1997, The Journal of Biological Chemistry.

[23]  G. Blobel,et al.  Kap104p: A Karyopherin Involved in the Nuclear Transport of Messenger RNA Binding Proteins , 1996, Science.

[24]  P. Bork,et al.  A Novel Class of RanGTP Binding Proteins , 1997, The Journal of cell biology.

[25]  S. Hegewisch-Becker,et al.  An optimized electroporation protocol applicable to a wide range of cell lines. , 1994, BioTechniques.

[26]  S. Sazer.  The search for the primary function of the Ran GTPase continues. , 1996, Trends in cell biology.

[27]  F. Melchior,et al.  A Small Ubiquitin-Related Polypeptide Involved in Targeting RanGAP1 to Nuclear Pore Complex Protein RanBP2 , 1997, Cell.

[28]  M. Ohtsubo,et al.  The RCC1 protein, a regulator for the onset of chromosome condensation locates in the nucleus and binds to DNA , 1989, The Journal of cell biology.

[29]  A. Lamond,et al.  Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities. , 1996, The EMBO journal.

[30]  F. Bischoff,et al.  Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

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

[32]  S. Adam,et al.  RanBP1 stabilizes the interaction of Ran with p97 nuclear protein import , 1996, The Journal of cell biology.

[33]  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.

[34]  G. Dreyfuss,et al.  A Novel Receptor-Mediated Nuclear Protein Import Pathway , 1996, Cell.

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

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

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

[38]  F. Bischoff,et al.  Identification of different roles for RanGDP and RanGTP in nuclear protein import. , 1996, The EMBO journal.

[39]  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.

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

[41]  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.

[42]  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.

[43]  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.

[44]  F. Bischoff,et al.  Dominant‐negative mutants of importin‐β block multiple pathways of import and export through the nuclear pore complex , 1997, The EMBO journal.

[45]  P. D’Eustachio,et al.  Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line , 1990, Molecular and cellular biology.

[46]  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.

[47]  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.

[48]  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.