GTP hydrolysis by Ran is required for nuclear envelope assembly

Nuclear formation in Xenopus egg extracts requires cytosol and is inhibited by GTP gamma S, indicating a requirement for GTPase activity. Nuclear enve lope (NE) vesicle fusion is extensively inhibited by GTP gamma S and two mu tant forms of the Ran GTPase, Q69L and T24N. Depletion of either Ran or RCC 1, the exchange factor for Ran, from the assembly reaction also inhibits th is step of NE formation. Ran depletion can be complemented by the addition of Ran loaded with either GTP or GDP but not with GTP gamma S. RCC1 depleti on is only complemented by RCC1 itself or by RanGTP. Thus, generation of Ra nGTP by RCC1 and GTP hydrolysis by Ran are both required for the extensive membrane fusion events that lead to NE formation.