Figure 3. Vacuolar LD consumption is dependent on AMPK and is not induced by rapamycin treatment.
(A) WT cells expressing GFP-Atg8 were placed in synthetic complete (SC) media containing 2% glucose (Gradual GR) or 0.4% glucose (Acute GR). Autophagic flux assay was performed as described in Figure 2F. (B) LD flux assay as described in Figure 2D was performed under similar conditions as in (A). (C) Autophagic flux assay was performed with 50 nM rapamycin under similar conditions as described in (A). (D) LD flux assay was performed with 50 nM rapamycin under similar conditions as described in (B). (E) LD flux assay was performed in WT, snf1Δ, and snf4Δ cells as described in Figure 2D. (F) Representative confocal images of cells in (E) with both Erg6-DsRed and Vph1-GFP (vacuole membrane marker) are shown. White arrowhead points to LD signal localized within the vacuole. Scale bar represents 5 μm. (G) Representative SXT images of WT and snf1Δ cells undergoing acute GR for day 1.5 (D1.5) are shown. Black arrows and white arrows point to LDs and autophagic bodies, respectively. Scale bar represents 1 μm. (H) Quantification of autophagic flux in WT, snf1Δ, and snf4Δ cells is shown as described in Figure 2F. Data are expressed as mean ± SD (n = 3). n indicates the number of experimental repetitions.
Figure 3—figure supplement 1. Snf1p is required for vacuolar LD consumption, cellular energy metabolism, and long-term survival during acute GR.
