PURIFICATION AND MASS-SPECTROMETRIC ANALYSIS OF ADP-RIBOSYLATION FACTOR PROTEINS FROM XENOPUS EGG CYTOSOL

The CTP analog GTP gamma S potently inhibits nuclear envelope assembly in cell-free Xenopus egg extracts. GTP gamma S does not affect vesicl e binding to chromatin but blocks Vesicle fusion. Fusion inhibition by GTP gamma S is mediated by a soluble factor, initially named GSF (GTP gamma S-dependent soluble factor), We previously showed that vesicles pretreated with GTP gamma S plus recombinant mammalian ARF1 were inhi bited 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 c ytosol. At all steps in the purification, fractions containing ARF, bu t no other fractions, showed GSF activity, The purified CSF was identi fied as Xenopus ARF by immunoblotting and peptide sequence analysis. R everse phase HPLC and mass spectrometry revealed that GSF contained at least three distinct ARF proteins, all of which copurified through th ree chromatography steps. The most abundant isoform was identified as ARF1 (62% of the total GSF), because its experimentally determined mas s 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. W e concluded that ARF is most likely the only soluble protein chat inhi bits nuclear vesicle fusion after pretreatment with GTP gamma S.