GTP hydrolysis by Ran is required for nuclear envelope assembly

Nuclear formation in Xenopus egg extracts requires cytosol and is inhibited by GTPγS, indicating a requirement for GTPase activity. Nuclear envelope (NE) vesicle fusion is extensively inhibited by GTPγS and two mutant forms of the Ran GTPase, Q69L and T24N. Depletion of either Ran or RCC1, the exchange factor for Ran, from the assembly reaction also inhibits this step of NE formation. Ran depletion can be complemented by the addition of Ran loaded with either GTP or GDP but not with GTPγS. RCC1 depletion is only complemented by RCC1 itself or by RanGTP. Thus, generation of RanGTP by RCC1 and GTP hydrolysis by Ran are both required for the extensive membrane fusion events that lead to NE formation.