Figure 6.

sec12-4 membranes fail to recruit Sec23p-GFP to tER sites at elevated temperatures in the presence of GTP, but not GTPγS. (A) Washed wild-type (CBY80) and sec12-4 (CBY558) semi-intact cell membranes, prepared from cultures grown at 20°C, were incubated with Sec23p-GFP cytosol (CBY1860) and Sar1p at 4°C for 30 min. DAPI was added, and a sample of each reaction was placed on a glass slide and imaged continuously at 23°C for 9 min. (B) Reactions were assembled and processed as in A, but GTP was replaced with GTPγS. (C) Plots showing total fluorescence intensity per semi-intact cell at different time points from the images obtained in A and B, analyzing five to 10 cells per time point. Note that GTPγS addition stabilizes Sec23p-GFP at tER sites in both wild-type and sec12-4 membranes throughout the time course.