Xenopus Ran-binding protein 1: molecular interactions and effects on nuclear assembly in Xenopus egg extracts.
Ran is a nuclear GTPase implicated in nucleocytoplasmic transport, the maintenance of nuclear structure, mRNA processing, and cell cycle regulation. By two-hybrid interaction in yeast, we have identified a Xenopus homologue of Ran-binding protein 1 (RanBP1). Xenopus RanBP1 interacts specifically with the GTP-bound form of Ran and forms complexes in Xenopus egg extracts with Ran, importin-beta/karyopherin-beta and importin-alpha/karyopherin-alpha, but not p10, p120/RanBP7, RanBP2 or other nucleoporins. These complexes may play roles in the recycling of Ran and importins/karyopherins during nucleocytoplasmic transport. Increased concentrations of RanBP1 stabilise an interaction between Ran and RCC1 in egg extracts, inhibiting the exchange activity of RCC1 towards Ran. Under these conditions, the assembly of nuclei from chromatin is dramatically affected: the nuclei do not assemble a lamina and become very small with homogeneously condensed chromatin. They fail to actively import proteins and do not undergo DNA replication. By field emission in-lens scanning electron microscopy, we show that these nuclei have an intact nuclear envelope containing pore complexes, but the envelope is highly convoluted. However, RanBP1 does not directly inhibit nuclear protein import in assembled nuclei. These results suggest that RCC1 and/or Ran have a function early in nuclear assembly that is disrupted by RanBP1.
Nicolás
F J
FJ
Zeneca Laboratory of Molecular and Cellular Biology, School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.
Zhang
C
C
Hughes
M
M
Goldberg
M W
MW
Watton
S J
SJ
Clarke
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014981:Xenopus; D029867:Xenopus Proteins; D020931:ran GTP-Binding Protein
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C499304:RCC1 protein, Xenopus; D029867:Xenopus Proteins; C091775:ran-binding protein 1; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein