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Vol. 8, Issue 10, 1955-1970, October 1997
Laboratory of Molecular Embryology, National Institute of Child
Health and Human Development, National Institutes of Health, Bethesda,
Maryland 20892-5431
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 by 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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