Fig. 7: Ate1-induced cell death involves mitochondrial outer membrane permeabilization.
From: Arginyltransferase1 drives a mitochondria-dependent program to induce cell death
A Growth of yeast cells (W303 strain, WT) carrying either the empty expression vectors (pYES2-URA3 and pBF339-TRP1 vectors), or the galactose-inducible mouse BAX (pBM272-pGAL-BAX-URA3) and yeast Ate1 (pYES2-pGAL-ATE1-GFP-Ura3) in the presence of constitutively expressing Bcl-xL (pBF339-ADH-BCLxL-TRP1) or the vector (pBF339-TRP1) was measured by a serial dilution growth assay on either plate containing 2% glucose or 2% galactose, where the expression of Ate1 or BAX is not induced or induced, respectively. Plates were incubated at 30 °C and images were taken after 3 days. B Representative Western blots displaying changes in the distribution of cytochrome c (Cyc) between mitochondria and cytosol in yeast cell where Ate1-6 × His was induced for expression (“+Ate1”) with 2% galactose in Ura-minus liquid media for 6 h at 30 °C. The separated cytosolic fraction and mitochondrial fraction were compensated by buffer to be equal volume before analysis. Alpha-tubulin and Porin (VDAC) were used as markers to display the purity of cytosolic and mitochondrial fractions, respectively. Pgk1 was used as an additional loading control. The quantification of the blot was based on n= 6; error bars represent S.E.M. C Growth of yeast cells (WT or aif1Δ) carrying either the empty expression pYES2 vector (“Vector”) or pYES-pGAL1:ATE1-GFP (“+Ate1-GFP”) was measured by a serial dilution growth assay on either plate containing 2% glucose or 2% galactose, where the expression of Ate1 is not induced or induced, respectively. Plates were incubated at 30 °C and images were taken after 3–5 days. D Similar to (C), except that WT, aif1Δ, ate1Δ¸ yca1Δ, nuc1Δ, and dnm11Δ) yeasts were used. E Similar to (C), except that WT and cyc3Δ yeasts were used. See also Suppl Fig. S1C to see the equal growth rate of the involved mutant yeast strains carrying the empty vector on galactose-containing media compared to the WT strain, if they were not already shown in this figure.
