Fig. 4.

The ArfGAP activity of GIT1 is blocked by an N-terminal tag. Purified bacterial Arf1 preloaded with [α³²P]GTP was mixed with the indicated amount of baculoviral-expressed GIT1-6xHis (C-terminal tag) or 6xHis-GIT1 (N-terminal tag) purified from Sf9 cells, and Arf-bound nucleotide (GTP and GDP) was separated by thin-layer chromatography. Whereas GIT1-6xHis catalyzes conversion of GTP to GDP, 6xHis-GIT1 does not, indicating that the N-terminal epitope tag interferes with the ability of GIT1 to recognize or interact with GTP-bound Arf. Similar effects have been noticed by using GIT1 tagged with GST or Flag tag (our unpublished observations). We thank Nicolas Vitale for performing this assay.