FIGURE 4.

Involvement of ESCRT machinery in autophagy-related vacuolar sorting of Ypk1 that is distinct from conventional endosomal sorting. A, Ypk1 expression in nitrogen-starved WT, vps27Δ, and vps23Δ cells. Logarithmically growing cells were starved of nitrogen (NS) for the times indicated. Expressions of Ypk1 and Pgk1 were analyzed by Western blotting as described in Fig. 1A. B and C, fluorescence microscopic analyses of GFP-Ypk1 localization in pep4Δvps27Δ cells. FM4-64 labeling and nitrogen starvation were performed as described in Fig. 2C. Arrows indicate accumulated GFP-Ypk1 at the perivacuolar structures where FM4-64 was strongly incorporated. Relative fluorescence intensities of GFP-Ypk1 (green) and FM4-64 (red) were plotted (C) as in Fig. 2D. Percentages noted below the images represent frequencies of the indicated phenotypes. D and E, fluorescence microscopic analyses of Tat2-EGFP localization in pep4Δvps27Δ cells. Logarithmically growing cells harboring pRS415-TAT2-EGFP were labeled with FM4-64 and starved of nitrogen as described in Fig. 2C. The arrows indicate focus localization of Tat2-EGFP, and the arrowheads point to FM4-64-incorporated structures. Relative fluorescence intensities of Tat2-EGFP (green) and FM4-64 (red) in D were plotted (E) as in C. Bar, 5 μm. a.u., arbitrary units; DIC, differential interference contrast.