FIGURE 5.

Ypk1 closely associates with autophagy-related structures in both pep4Δ and ESCRT mutant cells. A, fluorescence microscopic analyses of GFP-Atg8 localization in the nitrogen-starved pep4Δ and pep4Δvps27Δ cells. Images were obtained for cells harboring pRS415-GFP-ATG8 as described in Fig. 2C. Arrowheads point to intravacuolar localization in pep4Δ cells, whereas arrows indicate perivacuolar sites in pep4Δvps27Δ cells where GFP-Atg8 localized within FM4-64-incorporated structures. B–E, fluorescence microscopic analyses of GFP-Ypk1 and RFP-Atg8 localization in pep4Δ (B and C) and pep4Δvps27Δ (D and E) cells. Logarithmically growing cells harboring pRS415-GFP-YPK1 and pRS413-RFP-ATG8 were starved of nitrogen (NS) for 3 h in the presence of PMSF. Cell culture was terminated by NaF and NaN₃ treatment on ice as described under “Experimental Procedures,” and localization of GFP-Ypk1 and RFP-Atg8 fluorescence was immediately imaged after this treatment. Relative fluorescence intensities of GFP-Ypk1 (green) and RFP-Atg8 (red) in a and b were plotted (C and E, respectively) as in Fig. 2D. Percentages noted below the images represent frequencies of the indicated phenotypes. Bar, 5 μm. a.u., arbitrary units; DIC, differential interference contrast.