| Abstract
| - Arfophilin was first identified as a target protein for GTP-ARF5. The N-terminus of ARF5(amino acids 2−17), which is distinct from that of class I or class III ARFs, is essential for binding to theC-terminus of arfophilin (amino acids 612−756). This study using GST fusion proteins in pulldownexperiments in CHO-K1 cell lysates showed that, unexpectedly, ARF6 also bound to full-length arfophilinor the C-terminus of arfophilin (amino acids 612−756) in a GTP-dependent manner. Studies with ARF1/ARF6 chimeras further showed that the amino acid sequence of residues 37−80 of ARF6, which is differentfrom the corresponding sequences in class I and class II ARFs, was essential for binding to arfophilin.Both GTP-ARF5 and GTP-ARF6 bound to arfophilin in CHO-K1 cell lysates, while GTP-ARF1 did notbind. In contrast, all three forms of ARF bound to arfaptin 2, with ARF1 showing the strongest binding.Yeast two-hybrid studies with wild-type, dominant negative, and constitutively active forms of ARF1, -5,and -6 and with ARF1/ARF6 chimeras confirmed these results, except that constitutively active ARF6was autoactivating. Our findings suggest that both class II and III ARFs may influence the same cellularpathways through arfophilin as a common downstream effector.
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