ARF is not required for nuclear vesicle fusion or mitotic membrane disassembly in vitro: evidence for a non-ARF GTPase in fusion.

Abstract

Xenopus laevis egg extracts are a well-characterized system for studying nuclear envelope dynamics in vitro. ADP-ribosylation factor (ARF), a 21 kDa GTPase involved in vesicular transport, inhibits nuclear vesicle fusion in vitro when membranes are preincubated with ARF and GTP gamma S (Boman et al., Nature 358, 512-514 (1992)). To test the hypothesis that ARF was required for nuclear envelope assembly or disassembly, we examined these events in cytosol from which ARF was depleted by size 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 small. Further growth was not stimulated by adding ARF1, suggesting that fractionation removed other proteins required for pore complex assembly and nuclear growth. Nuclei assembled in ARF-depleted extracts, and rat liver nuclei, disassembled normally in mitotic ARF-depleted reactions. BFA, which inhibits ARF binding to membranes, had no effect on nuclear assembly or disassembly. We concluded that ARF is not essential for nuclear membrane dynamics. Nuclear vesicle fusion was still inhibited by GTP gamma S in ARF-depleted reactions and in reactions containing BFA, strongly suggesting that there is another unidentified GTPase that is either required for vesicle fusion or capable of inhibiting fusion in the presence of GTP gamma S.