FIG 5.

Gcn5-mediated acetylation on Pk lysine 188 may contribute to autophagy degradation of Pk. (A) Total protein lysates were extracted from the wild-type (WT) strain, three versions of PK-overexpressed strains in the WT background, PKOX in the gcn5Δ background, and WT, gcn5Δ, and atg1Δ (autophagy-deficient) strains expressing M2 (K188Q) version of PK. Total protein lysates were analyzed by immunoblotting with anti-GFP antibodies (rabbit; 1:5,000; Invitrogen Molecular Probes, catalog no. A6455). The presence of the free GFP band indicates cleavage between GFP and Pk protein, and likely degradation of fusion proteins. Coomassie blue staining of SDS-PAGE of total proteins served as a loading control. (B) Epifluorescence microscopy with PKOX, PK(M1)OX (K188R, unacetylated mimic) strain, and WT, gcn5Δ, and atg1Δ (autophagy-deficient) strains expressing M2 (K188Q) version of PK. Arrows denote spherical vacuoles and arrowheads indicate vesicular puncta or filamentous vacuoles. Bar = 5 μm.