The Gga proteins but not their Ub-binding GAT domain are required for Gap1 exit from the Golgi. sec14-3, gga1Δ gga2Δ, and sec14-3 gga1Δ gga2Δ cells were transformed with a centromeric vector encoding Gap1-GFP or Gap1^K9,16R-GFP or the latter plasmid additionally carrying the GGA2 or gga2^ΔGAT gene. Cells were treated as in Fig. 2. (A) Gap1-GFP stability was examined in total protein extracts prepared from cells collected before and at different times after the transfer to 37°C by immunoblotting with anti-GFP antibodies. (B) Gap1-GFP localization was examined by fluorescence microscopy before (not depicted) and at different times after the shift to 37°C. Am, ammonium. Bar, 5 µm.