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 pr ocessing, maintenance of structural integrity of nuclei, and cell cycl e control. RanBP1 is a highly conserved Ran guanine nucleotide dissoci ation inhibitor. We sought to use Xenopus egg extracts for the develop ment of an in vitro assay for RanBP1 activity in nuclear assembly, pro tein import, and DNA replication. Surprisingly, when we used anti-RanB P1 antibodies to immunodeplete RanBP1 from Xenopus egg extracts, we fo und that the extracts were also depleted of RCC1, Ran's guanine nucleo tide exchange factor, suggesting that these proteins form a stable com plex. In contrast to previous observations using extracts that had bee n depleted of RCC1 only, extracts lacking both RanBP1 and RCC1 (codepl eted 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 deple ted proteins caused abnormal nuclear assembly and inhibited nuclear tr ansport and DNA replication in a manner that could be rescued by furth er addition of RCC1 or RanBP1, respectively. Exogenous mutant Ran prot eins could partially rescue nuclear function in extracts without RanBP 1 or without RCC1, in a manner that was correlated with their nucleoti de binding state. These results suggest that little RanBP1 or RCC1 is required for nuclear assembly, nuclear import, or DNA replication in t he 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.