Gap1 exit from the Golgi is Ub independent and Vps1 dependent. Cells transformed with a centromeric vector encoding Gap1-GFP or Gap1^K9,16R-GFP were grown at 24°C in glucose-, glutamine-, and ammonium (Am)-containing medium to repress GAP1 gene expression. Synthesis of the permease was then induced at the restrictive temperature by transferring the cells to proline medium at 37°C. After 90 min, each culture was divided in two, glutamine and ammonium were added to one of the flasks, and the cells were incubated for 1 h more at 37°C. (A) Localization of Gap1-GFP and Gap1^K9,16R-GFP in sec14-3 cells was examined at different time points by fluorescence microscopy. GFP fluorescence is shown in the top rows, and Nomarski images are shown in the bottom rows of each pair of panels. (B) Localization of Gap1-GFP and Gap1^K9,16R-GFP in sec14-3 and vps1Δ sec14-3 cells at time 150 min was examined by subcellular fractionation in a sucrose gradient. (C) The level of Gap1-GFP protein was analyzed by immunoblotting in total protein extracts prepared from sec14-3 and sec7-1 cells collected at different times with anti-GFP antibodies. Bar, 5 µm.