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. Author manuscript; available in PMC: 2011 Apr 23.
Published in final edited form as: Mol Cell. 2010 Apr 23;38(2):250–264. doi: 10.1016/j.molcel.2010.02.033

Figure 4.

Figure 4

Overexpression of Pho85 induces autophagy and relieves the inhibitory effect of Sic1 overexpression on autophagy.

(A), (B) and (C) Overexpression of Pho85 induces autophagy. (A) and (B) Wild-type (W3030-1B), pcl5Δ pho80Δ (ZFY207), GAL1-PHO85 (ZFY209), pcl5Δ pho80Δ GAL1-PHO85 (ZFY208), atg1Δ pcl5Δ pho80Δ GAL1-PHO85 (ZFY217), clg1Δ pcl5Δ pho80Δ GAL1-PHO85 (ZFY246) and pcl5Δ pho80Δ rim15Δ GAL1-PHO85 (ZFY216) cells expressing GFP-Atg8 (pCU-GFP-AUT7(416)), were grown in SMD and shifted to SMGal for 12 h. In (A), cells that were grown in SMD (−) or SMGal (+) were collected and subjected to immunoblotting, as described in Figure 2A. In (B), cells that were grown in SMGal were analyzed by fluorescence microscopy as described in Supplemental Experimental Procedures. Bar, 2.5 μm.

(C) Wild type (ZFY202), pcl5Δ pho80Δ (ZFY206), GAL1-PHO85 (ZFY224), pcl5Δ pho80Δ GAL1-PHO85 (ZFY225) and atg1Δ pcl5Δ pho80Δ GAL1-PHO85 (ZFY227) cells were grown in YPD and shifted to YPGal for 12 h, then treated with rapamycin for 5 h and analyzed by the Pho8Δ60 assay. Values were normalized to the activity of wild-type cells with rapamycin treatment, which was set to 100%. Error bars indicate the SD of three independent experiments.

(D) In pcl5Δ pho80Δ cells, overexpression of Pho85 suppresses the autophagic defect resulting from Sic1 overexpression. pcl5Δ pho80Δ (ZFY207) and pcl5Δ pho80Δ GAL1-PHO85 (ZFY208) cells were analyzed by the GFP-Atg8 processing assay, as described in Figure 2A. See also Figure S4.