Identification of Arfophilin, a Target Protein for GTP-bound Class II ADP-ribosylation Factors

Abstract

Yeast two-hybrid screening of a human kidney cDNA library using the GTP-bound form of a class II ADP-ribosylation 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 GTP-bound 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.