Fig. 4. Dependence of GAP activity of AGAP2 for interaction with AP-1. (A-D) Effect of [Q71L]Arf1 on AGAP2 induced AP-1 redistribution. HeLa cells were transfected with FLAG-AGAP2 (B), [Q71L]Arf1-HA (C) or both (A,D) for 24 hours. Cells were stained for the AP-1 and FLAG tag (B,D), or HA-tag (C), or stained for both FLAG and HA tag (A). (E) Requirement of the conserved arginine for GAP activity. The catalytic core of AGAP2, PZA2, or its point mutant with the conserved arginine mutated to lysine, [R618K]PZA2, were expressed as GST-fusion proteins in E. coli and purified. The proteins were eluted from the beads with glutathione and dialyzed overnight against PBS with 1 mM dithiothreitol. Increasing concentrations of PZA2 or [R618K]PZA2 were titrated into the GAP assay as described in Materials and Methods. (F) Effect of GAP dead AGAP2 on AP-1 association with TGN. FLAG-tagged [R618K]AGAP2 was transfected into HeLa cells. Cells were fixed 24 hours after transfection and stained for AP-1 and the FLAG tag. Transfected cells were indicated by arrows.