A MUTANT FORM OF THE RAN TC4 PROTEIN DISRUPTS NUCLEAR FUNCTION IN XENOPUS-LAEVIS EGG EXTRACTS BY INHIBITING THE RCC1 PROTEIN, A REGULATOR OF CHROMOSOME CONDENSATION/

The Ran protein is a small GTPase that has been implicated in a large number of nuclear processes including transport, RNA processing and ce ll cycle checkpoint control, A similar spectrum of nuclear activities has been shown to require RCC1, the guanine nucleotide exchange factor (GEF) for Ran, We have used the Xenopus laevis egg extract system and in vitro assays of purified proteins to examine how Ran or RCC1 could be involved in these numerous processes, In these studies, we employe d mutant Ran proteins to perturb nuclear assembly and function, The ad dition of a bacterially expressed mutant form of Ran (T24N-Ran), which was predicted to be primarily in the GDP-bound state, profoundly disr upted nuclear assembly and DNA replication in extracts, We further exa mined the molecular mechanism by which T24N-Ran disrupts normal nuclea r activity and found that T24N-Ran binds tightly to the RCC1 protein w ithin the extract, resulting in its inactivation as a GEF. The capacit y of T24N-Ran-blocked interphase extracts to assemble nuclei from de-m embranated sperm chromatin and to replicate their DNA could be restore d by supplementing the extract with excess RCC1 and thereby providing excess GEF activity, Conversely, nuclear assembly and DNA replication were both rescued in extracts lacking RCC1 by the addition of high lev els of wild-type GTP-bound Ran protein, indicating that RCC1 does not have an essential function beyond its role as a GEF in interphase Xeno pus extracts.