Cloning of a carrot cDNA for a member of the family of ADP-ribosylation factors (ARFs) and characterization of the binding of nucleotides by its product after expression in E. coli.

A cDNA clone, designated DcArf1, was isolated from a cDNA library prepared from embryogenic cell clusters of Daucus carota and characterized. The cDNA (883 bp) contained an open reading frame that encoded a protein of 181 amino acid residues with significant homology to the ADP-ribosylation factors (ARFs) of animals and yeast. The DcArf1-related transcripts were detected at a nearly constant level during somatic embryogenesis. The DcArf1-encoded protein was expressed as a fusion protein in E. coli and was shown to have dithiothreitol-independent [alpha-32P]GTP-binding activity. This binding was completely prevented by a 100-fold molar excess of unlabeled GTP or GDP, while GMP, ANP, TNP, CNP and UNP had no effect on the binding. The binding of [35S]GTP gamma S to the recombinant DCARF1 protein was detected only in the presence of millimolar levels of both MgCl2 and dimyristoylphosphatidylcholine (DMPC). The amino acid sequence and the GTP-binding characteristics of the DCARF1 protein suggest that the DcArf1 cDNA encodes an ARF of carrot rather than a protein that only resembles such a factor, ADP-ribosylation factor-like protein (ARL).