Figure 4.

Autophagy genes are required for Acb1 secretion. (A) Yeast mutants deleted of atg genes 5, 7, 8, and 12 were incubated in starvation medium for 4 h. The buffer was tested in the bioassay. The data show an average of three experiments. (B) Cells described in A were Western blotted with anti-Acb1 antibody. Atg mutants do not show any change in the intracellular levels of Acb1. (C) Wild-type yeast, grh1Δ, and atg7Δ were transformed with a plasmid expressing GFP-Atg8 under the control of its endogenous promoter. The cells were starved as in A. Wild-type and grh1Δ strains show GFP fluorescence inside the vacuolar compartment. However, the atg7Δ strain lacked GFP fluorescence in the vacuole because of a defect in autophagy. Bar, 5 µm. (D) Wild-type, grh1Δ, and atg7Δ cells expressing GFP-Atg8 were starved for 4 h, and cell lysates were Western blotted with anti-GFP antibody to monitor the vacuolar proteolysis of GFP-Atg8. Wild type and grh1Δ, but not atg7Δ cells, showed a lower band with the apparent molecular weight of GFP alone caused by the proteolysis of GFP-Atg8 by vacuolar proteases (Shintani and Klionsky, 2004).