Figure 6.
Assembly of Atg proteins to the PAS is regulated in response to nutrient conditions. (A) Recruitment of Atg17 or Atg29 to the PAS is induced by starvation in atg11Δ cells. Atg29-GFP and Atg17-GFP were observed in wild-type or atg11Δ cells under both growth and rapamycin-treated (3 h) conditions. (B) Atg29 localization in response to nutrient conditions in wild-type or in atg11Δ cells. Cells grown in SD medium containing casamino acid supplemented with adenine, tryptophan, and uracil were shifted to SD(−N). Nitrogen-starved cells (−N, 2 h) were supplied with 2× SD medium containing casamino acid supplemented with adenine, tryptophan, and uracil, and then they were incubated for 10 min. (C) PAS recruitment of Atg8 in response to starvation. atg11Δatg8Δ cells (YYK865) harboring the CFP-ATG8 (pRS314) were observed as described in B. CFP-Atg8 and Atg29-YFP were colocalized in starved cells. (Noted that CFP signal was observed in the vacuole indicating the induction of autophagy.) (D) Accumulation of Atg17 and Atg29 to the PAS in atg1D211Aatg11Δ is starvation specific. The localization of Atg17-GFP and Atg29-GFP was analyzed as described in B. (E) Comparison of autophagic activity of wild-type cells and atg11Δ cells in response to cellular nutrient condition. Wild-type (OND88) or atg11Δ (YYK762) cells genomically expressing Pho8Δ60 protein were cultured in YEPD, and then they were shifted to SD(−N) at time 0. After SD(−N) for 4 h, nitrogen-starved cells were supplied with nutrients as described in B. After 1 or 2 h, cells were corrected and subjected to ALP assay. The bars represent the SD of three independent experiments. Bars (A–D), 5 μm.