The balance of RanBP1 and RCC1 is critical for nuclear assembly and nuclear transport.
Ran is a small GTPase that is essential for nuclear transport, mRNA processing, maintenance of structural integrity of nuclei, and cell cycle control. RanBP1 is a highly conserved Ran guanine nucleotide dissociation inhibitor. We sought to use Xenopus egg extracts for the development of an in vitro assay for RanBP1 activity in nuclear assembly, protein import, and DNA replication. Surprisingly, when we used anti-RanBP1 antibodies to immunodeplete RanBP1 from Xenopus egg extracts, we found that the extracts were also depleted of RCC1, Ran's guanine nucleotide exchange factor, suggesting that these proteins form a stable complex. In contrast to previous observations using extracts that had been depleted of RCC1 only, extracts lacking both RanBP1 and RCC1 (codepleted extracts) did not exhibit defects in assays of nuclear assembly, nuclear transport, or DNA replication. Addition of either recombinant RanBP1 or RCC1 to codepleted extracts to restore only one of the depleted proteins caused abnormal nuclear assembly and inhibited nuclear transport and DNA replication in a manner that could be rescued be further addition of RCC1 or RanBP1, respectively. Exogenous mutant Ran proteins could partially rescue nuclear function in extracts without RanBP1 or without RCC1, in a manner that was correlated with their nucleotide binding state. These results suggest that little RanBP1 or RCC1 is required for nuclear assembly, nuclear import, or DNA replication in the absence of the other protein. The results further suggest that the balance of GTP- and GDP-Ran is critical for proper nuclear assembly and function in vitro.
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Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-5431, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D018797:Cell Cycle Proteins; D002457:Cell Extracts / Q000235:genetics / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D002875:Chromosomes / Q000378:metabolism; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D005656:Fungal Proteins / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D015870:Gene Expression; D020662:Guanine Nucleotide Exchange Factors; D007399:Interphase; D008969:Molecular Sequence Data; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000378:metabolism; D020931:ran GTP-Binding Protein
D000906:Antibodies; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C499304:RCC1 protein, Xenopus; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C091775:ran-binding protein 1; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein