Fig 2: 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.

Fig 3: Kinases and phosphatases from the KinDel screen alter the abundance and electrophoretic mobility of Aly1: (A) Cells expressing the indicated Aly1 protein fused to GFP (pRS415-TEF1pr plasmids) in either WT or the noted gene deletion backgrounds were imaged by high-content confocal microscopy and whole-cell fluorescence of the cells was quantified using NIS.ai and Nikon GA3 software. The mean fluorescence intensity from whole-cell measurements (in arbitrary units, au) is plotted for each cell as a circle. The median fluorescence intensity is shown as a black line for each group and the error bars represent the 95% confidence interval. A yellow or black dashed line represents the median fluorescence intensity for Aly1 or Aly1PPXYless expressed in WT cells, respectively. Kruskal–Wallis statistical analysis with Dunn’s post hoc test was performed to compare the fluorescence distributions to the cognate WT. In black asterisks (*) or blue daggers (†), comparisons are made to Aly1 or Aly1PPXYless in WT cells, respectively (ns = not significant; three symbols have a p-value < 0.0005). (B) A subset of the fluorescent microscopy images acquired for the data presented in (A) are shown. CMAC is used to stain the vacuoles (shown in blue). Merge is overlaid with the transmitted light cell image as well as the fluorescence images. (C) Whole-cell extracts from the cells described in (A) were made, analyzed by SDS-PAGE and immunoblotting, and detected using an anti-GFP antibody. The REVERT total protein stain of the membrane is shown as a loading control. Molecular weights are shown on the left side in kDa.

Fig 4: Sit4 and Npr1 regulate Aly abundance but do not affect localization: (a) Aly1-GFP or (c) Aly2-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. All images are shown as equally adjusted from a single experiment. (b) or (d) The whole-cell fluorescence of cells imaged in (a) or (c), respectively, was determined using NIS.ai and Nikon GA3 software, and the fluorescence for each cell is plotted as a circle. The median fluorescence intensity is shown as a black line for each group and the error bars represent the 95% confidence interval. A yellow dashed line represents the median fluorescence intensity for Aly1 or Aly2, expressed in WT cells in panels b and d, respectively. Kruskal–Wallis statistical analysis with Dunn’s post hoc test was performed to compare the fluorescence distributions to the cognate WT. In black asterisks (*), statistical comparisons are made to Aly1 or Aly2 in WT cells for panels (b) and (d), respectively. In blue daggers (†), the indicated statistical comparisons are made (ns = not significant; three symbols = p-value < 0.0005). (e–h) Whole-cell extracts were made from the indicated strains expressing Alys as GFP-tagged proteins from the TEF1pr and then resolved by SDS-PAGE. Immunoblots were probed with anti-GFP antibody to examine Aly abundance and mobility. REVERT total protein stain is shown for each blot as a loading control (red). The percent of Aly protein, corrected for the loading control, for each lane is calculated relative to WT Aly in WT cells.