Isolation of an Amino-Terminal Deleted Recombinant ADP-Ribosylation Factor 1 in an Activated Nucleotide-Free State

ADP-ribosylation factors (ARFs) are ≈20-kDa guanine nucleotide-binding proteins that activate cholera toxin ADP-ribosyltransferase in vitro and participate in intracellular vesicular membrane trafficking. ARFs are activated when bound GDP is replaced by GTP and inactivated by hydrolysis of bound GTP to yield ARF-GDP. Usually, ARFs are isolated in an inactive GDP-bound state and require addition of GTP along with detergent or phospholipid for activity. Purified mutant recombinant ARF1 lacking the first 13 amino acids (rΔ13ARF1-P) stimulated cholera toxin activity essentially equally with or without added GTP (and phospholipid or detergent), at least in part due to the presence of bound nucleotides, which later were identified as GTP and GDP. Nucleotide-free rΔ13ARF1 (rΔ13ARF1-F), prepared by dialysis against 7 M urea, was active without added GTP in the absence of SDS but inactive without added GTP in its presence. Renaturation of rΔ13ARF1-F in the presence of GTP, ITP, or GDP yielded, respectively, rΔ13ARF1-GTP and rΔ13ARF1-ITP, which were active, and rΔ13ARF1-GDP, which was inactive. Effects of phospholipids and detergents on nucleotide exchangeability evaluated as effects on activity of rARF1 and rΔ13ARF1-F differed. With rΔ13ARF1-F, 100 μM ITP and 100 μM GTP were essentially equally effective in the presence of cardiolipin or SDS. The finding that rΔ13ARF1 differs from rARF1 in the effects of phospholipids and detergents on nucleotide binding is consistent with the conclusion that the ARF amino terminus plays an important role in nucleotide binding and its specificity as well as the molecular conformation and associated activity.