Small molecular weight GTP-binding proteins, including ADP- ribosylation factors (ARFs, cofactors for cholera toxin-dependent ADP-ribosylation), are localized in the secretory pathway and are thought to cycle between cytosol and membranes. Evidence for ARF function in this pathway includes the localization of ARF to Golgi-derived vesicles and also the detection of abnormal invertase secretion in ARF-1 mutant yeast. We hypothesized that ARF, which is purified primarily as a cytosolic protein, might shift to membranes after incubation with GTP or analogs. Pellet and supernatant fractions from rat pheochromocytoma (PC-12) cells were analyzed by Western blotting. ARF was predominantly cytosolic but increased in membranes during incubation of cell homogenates with 0.1 mM non-hydrolyzable GTP-analogues GTP-gamma-S, Gpp(NH)p, Gppp(CH2)p and ATP-gamma-S, but not with GTP, GDP, GDP-beta-S, GMP or ATP (30 degrees C, 90 min). Other non-hydrolyzable derivatives App(NH)p and App(CH2)p were inactive, and it appears that a nucleoside diphosphokinase) may generate GTP-gamma-S from ATP-gamma-S. Cytosolic ARF similarly associated with anionic phospholipids (phosphatidylserine, phosphatidylinositol and cardiolipin) in GTP-gamma-S-dependent fashion, but not with a cationic phospholipid, phosphatidylcholine. Nucleotide specificity for ARF binding to phospholipids was identical to that for membranes. The lack of activity with GTP in either case suggests that membrane/phospholipid binding is influenced by a GTPase, perhaps in the form of an ARF-specific GTPase-activating protein (GAP). ARF binding to phosphatidylserine was reversed by incubation of the phospholipid pellet in the absence of 0.1 mM GTP-gamma-S, and dissociation was coincident with transfer of cholera toxin stimulating activity to the corresponding supernatant fraction. Therefore, ARF exhibits several features of GTP-binding proteins thought to be involved with transport and secretion: 1) guanine nucleotide-sensitive localization to cytosol or membrane/phospholipid domains; 2) reversible association with membrane/phospholipid domains; 3) retention of functional (i.e., non-denatured) status throughout the cycle. The data are consistent with a function in which ARF cycles between soluble and membrane compartments in vivo.
