Direct and indirect association of the small GTPase ran with nuclear pore proteins and soluble transport factors: studies in Xenopus laevis egg extracts.
Ran is a small GTPase that is required for protein import, mRNA export, and the maintenance of nuclear structures. To gain a better understanding of Ran's role in the nucleus, we have sought to use Xenopus egg extracts for the purification and characterization of proteins from egg extracts bound with a high affinity to a glutathione-S-transferase-Ran fusion protein (GST-Ran). We found that GST-Ran associates specifically with at least 10 extract proteins. We determined the identifies of six Ran-interacting proteins (Rips), and found that they include RanBP2/Nup358, Nup153, Importin beta, hsc70, RCC1, and RanBP1. On the basis of peptide sequence, a seventh Rip (p88) seems to be similar but not identical to Fug1/RanGAP1, the mammalian Ran-GTPase-activating protein. Gel filtration analysis of endogenous extract proteins suggests that Importin beta acts as a primary GTP-Ran effector. Both Ran and Importin beta are coimmunoprecipitated by anti-p340RanBP2 antibodies in the presence of nonhydrolyzable GTP analogues, suggesting that Ran-Importin beta complexes interact with p340RanBP2. Two other Rips, p18 and p88, are coprecipitated with p340RanBP2 in a nucleotide-independent manner. Analysis of the Ran-GTPase pathway in Xenopus extracts allows the examination of interactions between Ran-associated proteins under conditions that resemble in vivo conditions more closely than in assays with purified components, and it thereby allows additional insights into the molecular mechanism of nuclear transport.
Saitoh
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Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-5430, USA.
Cooke
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CA
Burgess
W H
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Earnshaw
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WC
Dasso
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002850:Chromatography, Gel; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010063:Ovum / Q000737:chemistry / Q000378:metabolism*; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014981:Xenopus; D029867:Xenopus Proteins; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; C499304:RCC1 protein, Xenopus; C513268:RanGAP1 protein, Xenopus; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D028961:beta Karyopherins; C091775:ran-binding protein 1; C094493:ran-binding protein 2; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein