Purification and mass spectrometric analysis of ADP-ribosylation factor proteins from Xenopus egg cytosol

The GTP analog GTPγS potently inhibits nuclear envelope assembly in cell-free Xenopus egg extracts. GTPγS does not affect vesicle binding to chromatin but blocks vesicle fusion. Fusion inhibition by GTPγS is mediated by a soluble factor, initially named GSF (GTPγS-dependent soluble factor). We previously showed that vesicles pretreated with GTPγS plus recombinant mammalian ARF1 were inhibited for fusion, suggesting that 'GSF activity' was due to the ARF (ADP-ribosylation factor) family of small GTP-binding proteins. To ask if any soluble proteins other than ARF also inhibited vesicle fusion in the pretreatment assay, we purified GSF activity from Xenopus egg cytosol. At all steps in the purification, fractions containing ARF, but no other fractions, showed GSF activity. The purified GSF was identified as Xenopus ARF by immunoblotting and peptide sequence analysis. Reverse phase HPLC and mass spectrometry revealed that GSF contained at least three distinct ARF proteins, all of which copurified through three chromatography steps. The most abundant isoform was identified as ARF1 (62% of the total GSF), because its experimentally determined mass of 20 791 Da matched within experimental error that predicted by the sequence of the Xenopus ARF1 cDNA, which is reported here. The second most abundant isoform (25% of GSF activity) was identified as ARF3. We concluded that ARF is most likely the only soluble protein that inhibits nuclear vesicle fusion alter pretreatment with GTPγS.