Biochemical and Genetic Analysis of the Mitochondrial Response of Yeast to BAX and BCL-X_L

Abstract

The BCL-2 family includes both proapoptotic (e.g., BAX and BAK) and antiapoptotic (e.g., BCL-2 and BCL-X_L) molecules. The cell death-regulating activity of BCL-2 members appears to depend on their ability to modulate mitochondrial function, which may include regulation of the mitochondrial permeability transition pore (PTP). We examined the function of BAX and BCL-X_L using genetic and biochemical approaches in budding yeast because studies with yeast suggest that BCL-2 family members act upon highly conserved mitochondrial components. In this study we found that in wild-type yeast, BAX induced hyperpolarization of mitochondria, production of reactive oxygen species, growth arrest, and cell death; however, cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Coexpression of BCL-X_L prevented all BAX-mediated responses. We also assessed the function of BCL-X_L and BAX in the same strain of Saccharomyces cerevisiae with deletions of selected mitochondrial proteins that have been implicated in the function of BCL-2 family members. BAX-induced growth arrest was independent of the tested mitochondrial components, including voltage-dependent anion channel (VDAC), the catalytic β subunit or the δ subunit of the F₀F₁-ATP synthase, mitochondrial cyclophilin, cytochrome c, and proteins encoded by the mitochondrial genome as revealed by [rho⁰] cells. In contrast, actual cell killing was dependent upon select mitochondrial components including the β subunit of ATP synthase and mitochondrial genome-encoded proteins but not VDAC. The BCL-X_L protection from either BAX-induced growth arrest or cell killing proved to be independent of mitochondrial components. Thus, BAX induces two cellular processes in yeast which can each be abrogated by BCL-X_L: cell arrest, which does not require aspects of mitochondrial biochemistry, and cell killing, which does.

ACKNOWLEDGMENTS

A.G. was supported by a fellowship from the Leukemia Society of America, and this work was supported by grants from the NIH to S.J.K. and M.F.