FUNCTIONAL INTERACTION OF ADP-RIBOSYLATION FACTOR-1 WITH PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE

The relationship between ADP-ribosylation factor (Arf) 1 and phosphoin ositides, which have been independently implicated as regulators of me mbrane traffic, was examined. Because both Arf-dependent phospholipase D and Arf1 GTPase-activating protein (GAP) require phosphatidylinosit ol 4,5-bisphosphate (PIP2), Arf1 complexed with PIP2 has been proposed to interact with target proteins. This hypothesis was tested using Ar f1 GAP as a model system. Arf1 was shown to bind 60 PIP2 in Triton X-1 00 micelles with a K-d of 45 +/- 13 mu M. Arf1 also bound phosphatidic acid but with 10-fold lower affinity. PIP2 binding was specifically d isrupted by mutating lysines 15, 16, and 181 and arginine 178 to leuci nes, Decreased PIP2 binding resulted in an increased EC(50) of PIP2 fo r activation of Arf GAP. None of the mutations that decreased PIP2 bin ding affected binding to or activation of GAP by phosphatidic acid, wh ich acts at a functionally distinct site. These data support the hypot hesis that PIP2 binding to Arf1 promotes interaction with Arf GAP. The implications of lipid directed protein protein interactions for membr ane traffic are discussed.