ARF IS NOT REQUIRED FOR NUCLEAR VESICLE FUSION OR MITOTIC MEMBRANE DISASSEMBLY IN-VITRO - EVIDENCE FOR A NON-ARF GTPASE IN FUSION

Xenopus laevis egg extracts are a well-characterized system for studyi ng nuclear envelope dynamics in vitro, ADP-ribosylation factor (ARF), a 21 kDa GTPase involved in vesicular transport, inhibits nuclear vesi cle fusion in vitro when membranes are preincubated with ARP and GTP g amma S (Boman et al., Nature 358, 512-514 (1992)), To test the hypothe sis that ARF was required for nuclear envelope assembly or disassembly , we examined these events in cytosol from which ARF was depleted by s ize fractionation or inhibited with brefeldin A (BFA), In ARF-depleted extracts, vesicles bound chromatin and fused to enclose the chromatin , but the resulting enclosed nuclei lacked pore complexes and remained smalt, Further growth was not stimulated by adding ARF1, suggesting t hat fractionation removed other proteins required for pore complex ass embly and nuclear growth, Nuclei assembled in ARF-depleted extracts, a nd rat liver nuclei, disassembled normally in mitotic ARF-depleted rea ctions, BFA, which inhibits ARF binding to membranes, had no effect on nuclear assembly or disassembly, We concluded that ARF is not essenti al for nuclear membrane dynamics, Nuclear vesicle fusion was still inh ibited by GTP gamma S in ARF-depleted reactions and in reactions conta ining BFA, strongly suggesting that there is another unidentified GTPa se that is either required for vesicle fusion or capable of inhibiting fusion in the presence of GTP gamma S.