GTP-BINDING PROTEIN STRUCTURE/FUNCTION STUDIES

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ADP-ribosylation factors (ARFs) are multi-functional, multi-domain, proteins that regulate vesicular trafficking events in the ER and Golgi as well as at the plasma membrane by associating with a lipid membrane after becoming activated by binding GTP. This association, facilitates ARF interaction with effectors [e.g., the vesicular coat protein beta-COP found associated with type I Golgi-derived vesicles, the G-protein betagamma, or a specific isoform of phospholipase D (PLD)] and to recruit to membranes, proteins that contribute to budding and vesicle formation. ARFs were identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity. We had shown using chimeric proteins derived from ARF1 and human ARF-like protein (ARL1), which activates PLD only minimally, that the PLD activation site lies within amino acids 1-73 of ARF1, and the CTA activation site lies within residues 73-181. To define these sites more precisely, amino-terminally truncated ARFs, chimeric ARFs, and ARF point mutants were constructed. Amino-terminal truncation of ARF1 to an extent that significantly reduced its ability to activate PLD, did not impair its ability to bind GTP or to enhance the activity of cholera toxin in vitro. The chimeric protein ARF28ARL (the N-terminal 28 amino acids of ARF1 linked to the C-terminal 153 amino acids of ARL1) had less ability to activate PLD than did ARF50ARL. Thus, amino acids between 28 and 50 that differ in ARF and ARL, appear to be involved in the activation of PLD. Specifically, mutation of Lys38 in ARF to Gln, the corresponding amino acid in ARL, produced ARF1(K38Q) with diminished PLD activation. The corresponding ARL mutant, ARL1(Q38K), activated PLD much more effectively than did ARL itself. Data from the chimeras also narrowed the CTA activation domain to a region between amino acids 73 and 139. Overall, it appears that at least two different regions of the linear sequence of ARF are involved in the activation of PLD, and a third is involved in activation of cholera toxin ADP-ribosyltransferase. Thus, ARF is a multi-domain protein involved in multiple effector interactions.