Figure 7.
The interaction between Arf6-GTP and the Grp PH domain is direct and requires phosphoinositide. (A) Fluorescence scan of 20 μg purified YFP Grp1 PH domain or its mutants R284C, I307E, or K340L (B) Twenty micrograms of purified YFP Grp1 PH domain or its mutants was incubated with 20 μg purified GST-Arf6 T27N or GST-Arf6 Q67L immobilized on glutathione Sepharose beads in the presence (+) or absence of 30 nM Ins(1,3,4,5)P4. Bound proteins were resolved by SDS-PAGE, scanned for fluorescence of the YFP PH domains, and subsequently stained with Coomassie to visualize the GST-Arf6. The wt YFP Grp1 PH domain bound to GST-Arf6Q67L only in the presence of Ins (1,3,4,5)P4.