Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain.
Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor (ARF)-bound GDP for GTP by a Golgi-associated guanine nucleotide-exchange protein (GEP) [Helms, J.B. & Rothman, J.E. (1992) Nature (London) 360, 352-354; Donaldson, J.G., Finazzi, D. & Klausner, R.D. (1992) Nature (London) 360, 350-352]. Cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, it was no longer BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 305-309]. We describe here purification from bovine brain cytosol of a BFA-inhibited GEP. After chromatography on DEAE-Sephacel, hydroxylapatite, and Mono Q and precipitation at pH 5.8, GEP was eluted from Superose 6 as a large molecular weight complex at the position of thyroglobulin (approximately 670 kDa). After SDS/PAGE of samples from column fractions, silver-stained protein bands of approximately 190 and 200 kDa correlated with activity. BFA-inhibited GEP activity of the 200-kDa protein was demonstrated following electroelution from the gel and renaturation by dialysis. Four tryptic peptides from the 200-kDa protein had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae (total of 51 amino acids), consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7 that plays a similar role in cellular vesicular transport and Sec7 may be a GEP for one or more yeast ARFs.
Pulmonary Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
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Morinaga
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Tsai
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Moss
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Vaughan
D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D020126:Brefeldin A; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003517:Cyclopentanes / Q000494:pharmacology; D017886:Durapatite; D005656:Fungal Proteins / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D007700:Kinetics; D008969:Molecular Sequence Data; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D003517:Cyclopentanes; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D011500:Protein Synthesis Inhibitors; C066755:Sec7 guanine nucleotide exchange factors; D020126:Brefeldin A; D017886:Durapatite; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors