FIG. 4.

Interaction of 3A mutants with GBF1 and effects on GBF1 dynamics. (A) Interaction of wt 3A, 3A-REIKI, 3A-ins16S, 3A-PPP, 3A-LL, and 3A-VREY with the N-terminal part of GBF1 in yeast two-hybrid analysis. The upper, middle, and lower panels show growth of yeast on nonselective medium (leucine- and tryptophan-deficient medium [NS]), selective medium lacking histidine (−His), and selective medium lacking adenine (−Ade), respectively. (B) Interaction of wt 3A, 3A-REIKI, 3A-ins16S, 3A-PPP, 3A-LL, and 3A-VREY with the N-terminal part of GBF1 in mammalian two-hybrid analysis. The firefly luciferase activities measured at 48 h posttransfection are depicted. The activity measured with wt 3A and the GBF1 N terminus was set at 100%. (C) Dynamics of YFP-GBF1. FRAP traces were done for cells expressing YFP-GBF1 together with CFP fusion proteins of wt 3A, 3A-ins16S, 3A-PPP, 3A-LL, and 3A-VDSE. The traces show the average recoveries (n ≥ 10 cells) for at least two independent experiments. The fluorescence intensity before bleaching was normalized to 1, and the fluorescence intensity directly after bleaching was normalized to 0. The fluorescence intensity was corrected for bleaching of the cell during imaging and for background fluorescence.