FIGURE 3.

Ypk1 is a novel and selective substrate for nitrogen starvation-specific proteolysis requiring autophagy system. A, fluorescence microscopic analyses of GFP-Ypk1 in PMSF-treated pep4Δatg1Δ cells. Logarithmically growing pep4Δatg1Δ cells harboring pRS415-GFP-YPK1 were pulse-chased with FM4-64, starved of nitrogen, and examined by fluorescence microscopy for GFP-Ypk1 and FM4-64 as described in Fig. 2C. Percentages noted below the images represent frequencies of the indicated phenotypes. Bar, 5 μm. B and C, time course analysis of Ypk1 expression in nitrogen-starved WT, atg1Δ (B), atg5Δ (C), and atg7Δ (C) cells. Logarithmically growing cells were starved of nitrogen (NS) for the times indicated. Western blot analysis was performed as described in Fig. 1A. D, comparison of the degradation kinetics between Ypk1 and Ald6-GFP. Total cell lysates were prepared from ALD6-GFP cells starved of nitrogen for the indicated times and analyzed by Western blotting. The expression of Ald6-GFP was detected using anti-GFP antibody. Relative expressions of Ald6-GFP and Ypk1 were normalized by Pgk1 signal intensities and plotted on a line graph as means of three independent experiments. Error bars indicate S.E. Asterisks show statistical significance at p < 0.05, determined using Student's t test. E, comparison of degradation kinetics Ypk1 between nitrogen-starved or rapamycin-treated WT cells harboring pRS316-GFP-ATG8. Logarithmically growing cells in SD medium supplemented with auxotrophic nutrients were shifted to SD-N medium or treated with 200 ng/ml rapamycin. Cells were harvested at the indicated times, and Western blots against Ypk1 and Pgk1 were performed as described in Fig. 1A. Autophagic progression was monitored by GFP-Atg8 processing by Western blotting using an antibody against GFP. Asterisks indicate nonspecific signals. F, effect of various types of starvation on Ypk1 expression. Logarithmically growing cells harboring pRS316-GFP-ATG8 in SD medium supplemented with auxotrophic nutrients were shifted to SD-N medium, SD-P/SD-S medium with auxotrophic nutrients, or SD medium lacking auxotrophic nutrients. Cells were harvested at the indicated time point, and total cell lysates were analyzed by Western blotting as described in E. DIC, differential interference contrast.