Yeast two-hybrid screening of a human kidney cDNA library using the GTP-bound form of a class II ADPribosylation factor (ARF5) identified a novel ARF5-binding protein with a calculated molecular mass of 82.4 kDa, which was named arfophilin. Northern hybridization analysis showed high level arfophilin mRNA expression in human heart and skeletal muscle. Arfophilin bound only to the active, GTP-bound form of ARF5 and did not bind to GTP-ARF3, which is a class I ARF. The N terminus of ARF5 (1-17 amino acids) was essential for binding to arfophilin. The GTP-bound form of ARF5 with amino acid residues in the N terminus mutated to those in ARF4 (another class II ARF) also bound to arfophilin, suggesting it is a target protein for GTPbound forms of class II ARFs. The binding site for ARF on arfophilin was localized to the C terminus (residues 612-756), which contains putative coiled-coil structures. Recombinant arfophilin overexpressed in CHO-K1 cells was localized in the cytosol and translocated to a membrane fraction in association with GTP-bound ARF5. ARF5 containing the N terminus of ARF3 did not promote translocation indicating that class II ARFs are specific carriers for arfophilin.The ADP-ribosylation factors (ARFs) 1 were originally identified as cofactors for cholera toxin-catalyzed ADP-ribosylation of G␣ s , the ␣-subunit of the G protein that stimulates adenylate cyclase. ARFs have now been associated with intracellular membrane trafficking events such as recruitment of clathrincoated vesicle adaptor protein or coatomer protein to Golgi membranes, and receptor-mediated endocytosis (1, 2). As a subfamily of the Ras-related small GTP-binding proteins, ARF proteins transmit signals to downstream effectors in a guanine nucleotide-dependent manner. Guanine nucleotide exchange factors (GEFs) act on ARFs to catalyze replacement of bound GDP with GTP, converting them to the active GTP-bound form, while GTPase-activating proteins hydrolyze the bound GTP, converting them to the inactive GDP-bound form.Six mammalian ARF genes have been cloned. Based on phylogenetic analysis, deduced amino acid sequence, protein size, and gene structure, ARFs can be divided into three classes:class I (ARF1, ARF2, and ARF3), class II (ARF4 and ARF5), and class III (ARF6) (3). ARF6 is found in plasma membranes or endosomes, and is involved in peripheral vesicle trafficking such as endocytosis and exocytosis (4 -6). GDP-bound forms of class I and class II ARFs are mainly found in the cytosol (7). However, GTP-bound forms of class I and II ARFs can be associated with Golgi, endoplasmic reticulum, and endosomes (7,8). Class I and class II ARFs appear to be similar in both their cellular localization and functions. Both ARF1 and ARF5 are equally effective in promoting the recruitment of the AP-1 adaptor complex in Golgi (9). Peptides corresponding to the N-terminal 17 amino acids of ARF1 or ARF4 inhibit endoplasmic reticulum to Golgi vesicle transport (10), and expression of either ARF1 or ARF4 genes corrects the impairment of secretion in yea...