Figure 10.

TORC1-Sit4-Npr1 regulation of Aly-mediated trafficking of Git1: (a) Git1-GFP was expressed from the TEF1pr in either WT cells or those lacking the gene indicated and imaged by high-content fluorescence microscopy. CMAC is used to stain the vacuoles (shown in blue) and trypan blue (shown in red) is used to mark the cell wall. (b) The PM and vacuolar fluorescence intensities from the cells depicted in (a) were quantified using NIS.ai and Nikon GA3 software and the distributions of the PM/vacuole fluorescence ratios in arbitrary units (a.u.) were plotted as scatter plots. The horizonal midline in black represents the median and the 95% confidence interval is represented by the error bars. Yellow and grey dashed lines are used as references to indicate the median ratio for WT or 9Arr∆ cells, respectively. Kruskal–Wallis statistical analysis with Dunn’s post hoc test was performed to compare the fluorescence distributions to either WT or 9Arr∆ cells. In black asterisks (*), statistical comparisons are made to WT cells. In blue daggers (†), statistical comparisons are made to 9Arr∆ cells. (ns = not significant; three symbols = p-value < 0.0005). (c) Whole-cell extracts from cells expressing Git1-GFP from the TEF1 promoter were resolved by SDS-PAGE and immunoblotted. Anti-GFP antibody was used to detect Git1-GFP and REVERT total protein stain was used as a loading control. Blot shown is representative of 2 replicate experiments. Molecular weights are shown on the left side in kDa. The ratio of GFP breakdown product (represents the vacuolar pool of GFP) over Git1-GFP (represents the pool of Git1 outside of the vacuole) for each lane is presented. (d) Model of TORC1-Sit4-Npr1 regulation of Aly-mediated trafficking of Git1.