Figure 1.

ARF-binding region of GGA proteins and effects of mutations. (A) Comparison of ARF-binding domains from yeast and human GGA proteins. GGA proteins have four domains, with the highest identity between yeast and human GGA proteins in the ARF-binding domain. Residues identical in at least three proteins are highlighted in red. Dashes indicate gaps in the alignment. The numbers indicate the first and last residues shown for each GGA protein. Asterisks indicate mutated residues. (B) Loss of ARF binding by two-hybrid analysis. Gal4AD fusions of wild-type and mutant GGA proteins were assayed for binding to Gal4BD fusions of activated ARF in the two-hybrid assay. Serial 10-fold dilutions of each strain were replica plated on SD plates lacking tryptophan and leucine (−trp-leu) and SD plates lacking histidine, tryptophan, and leucine with 25 mM 3AT. Growth on −trp-leu shows that all strains are viable. Interaction is positive when colonies grow on 3AT and turn blue in X-gal assays. Scores in the right-hand column are defined in MATERIALS AND METHODS. (C) Loss of Arf binding by using affinity chromatography. Increasing concentrations of Arf1p·GDP or Arf1p·GTPγS (as indicated) were incubated with GST fusion proteins immobilized on glutathione agarose beads: GST-Gga2p (lanes 1–6), GST-Gga2p^I207N (lanes 7–12), and GST only (lanes 13–14). Retained proteins were eluted and analyzed by Coomassie staining to visualize the GST fusion proteins (bottom) or by immunoblotting to detect Arf1p (top). Arf1p was retained by GST-Gga2p, but not by mutant GST-Gga2p^I207N or GST only.