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 ESCHERICHIA-COLI

A cDNA clone, designated DcArf1, was isolated from a cDNA library prep ared from embryogenic cell clusters of Daucus carota and characterized . The cDNA (883 bp) contained an open reading frame that encoded a pro tein of 181 amino acid residues with significant homology to the ADP-r ibosylation factors (ARFs) of animals and yeast. The DcArf1-related tr anscripts were detected at a nearly constant level during somatic embr yogenesis. The DcArf1-encoded protein was expressed as a fusion protei n in E. coli and was shown to have dithiothreitol-independent [alpha-P -32]GTP-binding activity. This binding was completely prevented by a 1 00-fold molar excess of unlabeled GTP or GDP, while GMP, ANP, TNP, CNP and UNP had no effect on the binding. The binding of [S-35]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 car rot rather than a protein that only resembles such a factor, ADP-ribos ylation factor-like protein (ARL).