FIGURE 3:

Chs6(ΔTPR1-4) and Chs6(ΔTPR5) cannot be efficiently recruited to the Golgi. (A) Chs6(ΔTPR1-4)-9myc and Chs6(ΔTPR5)-9ymc display a reduced membrane association in cell lysates. Ten OD₆₀₀ of cells were spheroplasted, regenerated, and subsequently lysed in hypotonic buffer. Lysates were cleared of unbroken cells and subjected to differential centrifugation at 4°C. TCL, total cell lysate; P13, 13,000 × g pellet; S100, 100,000 × g supernatant; P100, 100,000 × g pellet; PM, plasma membrane. All constructs were chromosomally expressed under the native CHS6 promoter. (B) TPR mutants show inefficient Golgi localization in vivo. Chs6p‑3GFP and Chs6(ΔTPR1-4)-3GFP were chromosomally expressed under the native CHS6 promoter. Chs6(ΔTPR1-4)-3GFP was almost entirely cytoplasmic and showed no association with Golgi membranes. Chs6(ΔTPR5)-3GFP, expressed at a level similar to wild-type Chs6p using an inducible methionine promoter, was partially Golgi localized (arrowheads). Scale bar, 5 μm. (C) Quantification of results in B. Graph shows a total of three experiments. At least 95 cells were scored per experiment; only budded cells were used for scoring; only GFP dots overlapping with Sec7-dsRed were considered as TGN. Drawn with Origin software (OriginLab, Northampton, MA). Lower whisker represents 5th percentile; box represents 25th, 50th, and 75th percentiles; upper whisker represents 95th percentile.