ADP-ribosylation factors (ARFs) are approximately 20 kDa proteins that act as GTP-dependent allosteric activators of cholera toxin ADP-ribosyl transferase activity. ARFs comprise an extensive multigene family with a very high degree of sequence conservation, and an unusual evolutionary relationship to other families of signal-transducing guanine nucleotide-binding proteins. One explanation for the pattern of common properties and amino acid sequence identities found between the G alpha subunits and ARFs and between ras-like proteins and ARFs is that these three families of genes evolved from a common ancestor gene that most closely resembled ARF. To investigate the evolutionary origin of the ARFs, and as a precursor to planned developmental , cell lineage, and gene deletion studies, we isolated and sequenced ARF genes from simple eukaryotic systems, including Giardia lamblia, Drosophila and C. elegans, and studied ARF biochemical and immunological activity from these organisms. A Drosophila ARF gene had >94% amino acid identity to mammalian ARFs 1-3 (Class 1). Two genes from C. elegans differed greatly in degree of sequence identities; C. elegans ARF-a was >94% identical in amino acid sequence to Class 1 ARFs, whereas C. elegans ARF-b was <45% identical to any of the known ARFs. A single ARF gene isolated from two different Giardia lamblia strains had 63-72% amino acid sequence identity to mammalian ARFs. As Giardia lamblia is believed to have diverged from the rest of the eukaryotes before the endosymbiotic events leading to mitochondria, it is believed to represent the most ancient living eukaryotic organism. ARF is believed to be the first GTP-binding protein of either approximately 20 kDa or approximately 40 kDa classes found in a protozoan. Homogenates of Giardia lamblia, and C. elegans, stimulated cholera toxin-catalyzed ADP-ribosylation, and an antibody against bovine sARF-II reacted with two distinct protein bands in C. elegans soluble fractions. The ubiquitous expression of ARF in eukaryotic tissues suggests at least one and possibly several essential roles for ARF in cellular physiology that may be elucidated through combinations of cellular, molecular, and evolutionary biological approaches.
