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. 2017 May 22;114(23):E4539–E4548. doi: 10.1073/pnas.1701030114

Fig. 5.

Fig. 5.

Mutation of the cationic triad in Vac8p causes disruption of the NVJ and CVT pathway. (A) The Vac8p-triple mutant fails to support cytoplasm-to-vacuole transport of Ape1p-EGFP upon starvation. The vacuoles of wild-type, vac8Δ, and vac8-triple mutant cells expressing Ape1p-EGFP were labeled with FM4/64 in YPD media for 2 h at 30 °C. After free dye was removed, cells were resuspended in nitrogen starvation medium (SD-N) and allowed to grow for 2 h at 30 °C. GFP signals for Ape1p and FM4/64 signal in vacuoles were then analyzed by fluorescence microscopy. Representative images from each cell type are shown. (Magnification: 100×.) (B) Graph showing quantitative data for cytoplasmic and vacuolar Ape1p-EGFP obtained as described in A. (C) The Vac8p-triple mutant does not support the formation of a tight junction between the nucleus and the vacuole. Experiments were performed as in A without starvation, and GFP fluorescence for Nvj1p was analyzed by fluorescence microscopy. (Magnification: 100×.) DIC, differential interference contrast.