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Fig. 5. Characterization of the VAN3 protein. (A,B) ARF–GAP activity of recombinant VAN3 proteins in vitro. GAP activity was assessed by the hydrolysis of GTP bound to the yeast ARF1 protein. (A) Concentration dependence of VAN3. Recombinant VAN3 protein was serially diluted, and incubated with 1 µM [-³²P]GTP-loaded ARF protein at 30°C for 10 minutes. (B) Time-course of ARF–GAP activity of VAN3. Recombinant VAN3 (1 µM) was incubated with 1 µM [-³²P]GTP-loaded ARF1 protein at 30°C. Aliquots were withdrawn at the indicated times. (C) Fat western blots of phospholipids probed with recombinant VAN3. Phospholipids are indicated above each blot, and the amount of lipid spotted onto the nitrocellulose is shown to the left of each row of lipid. The blot was incubated with 0.5 µg/ml GST-tagged recombinant type V VAN3. PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI-4-P, phosphatidylinositol 4-monophosphate; PI-4,5-P2, phosphatidylinositol 4,5-bisphosphate. (D) Homodimerization of VAN3 through the BAR domain. Constructs including full-length VAN3 (FLVAN3) and seven types of truncated VAN3 were fused to GAL4 AD in pGADT7, and those containing FLVAN3 and type I VAN3 were fused to GAL4 DNA-BD in pGBKT7. Proteins are represented by bars; motifs or domains within proteins are indicated by different colors. Circles show protein–protein interactions. Crosses show no interaction.

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