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 ␤ subunit or the ␦ subunit of the F 0 F 1 -ATP synthase, mitochondrial cyclophilin, cytochrome c, and proteins encoded by the mitochondrial genome as revealed by [rho 0 ] cells. In contrast, actual cell killing was dependent upon select mitochondrial components including the ␤ 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.

Bax and Bcl-xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator

“…Consistent with the results of the isolated mitochondria, cytochrome c release from the mitochondria was observed when Bax was expressed in wild type, but not VDAC1-de®cient, yeast cells ( Figure 4f). The small magnitude of Bax-induced cytochrome c release observed in yeast cells, which is consistent with recent reports (Roucou et al, 2000;Gross et al, 2000), as opposed to the larger magnitude of cytochrome c release occurring in isolated mitochondria, could be due to di erences in the amount of Bax acting on the mitochondria or Bax-induced cell death, probably independent of cytochrome c release, that occurs before Bax-mediated increases of mitochondrial membrane permeability in cells. Although yeast cells carry two VDACs, VDAC1 and VDAC2, VDAC2 is known to make only a small contribution to the modulation of membrane permeability to metabolites (Lee et al, 1998).…”

“…Yeast cells have been used to study the function of Bcl-2 family of proteins (Greenhalf et al, 1996;Matsuyama et al, 1998;Priault et al, 1999a,b;Shimziu et al, 1999;Gross et al, 2000;Harris et al, 2000). ANT-de®cient yeast was shown to be resistant to Baxinduced cell death (Marzo et al, 1998) and this observation was taken as genetic evidence that ANT is a functional target of Bax, although the controversial results were also reported (Priault et al, 1999b).…”

“…ANT-de®cient yeast was shown to be resistant to Baxinduced cell death (Marzo et al, 1998) and this observation was taken as genetic evidence that ANT is a functional target of Bax, although the controversial results were also reported (Priault et al, 1999b). It has been shown that Bax does not kill r 7 yeast or ATP4 (a component of F1F0ATPase)-de®cient yeast Gross et al, 2000;Harris et al, 2000), and that addition of ATP enhances Bax-induced mitochondrial changes (Priault et al, 1999a), and respiration was required for Bax-induced cytochrome c release in isolated mitochondria . These results suggest that ATP (or oxidative phosphorylation) seems to be important for the cytotoxicity of Bax in yeast cells.…”

“…As for VDAC, there were controversial reports that VDAC-de®cient yeast cells are partially resistant (Gross et al, 2000) or more sensitive to Bax-induced death (Harris et al, 2000). Accumulating evidence, however, suggests that the mechanism by which Bax kills yeast is substantially di erent from apoptotic mechanism activated by Bax in mammalian cells Gross et al, 2000). For Figure 6 Protective e ect of Bcl-x L on mitochondria is independent of other Bcl-2 family members and ANT.…”

“…Addition of 100 mM atractyloside to mitochondria before addition of rBax is indicated as the solid column chondrial changes, we investigated whether ANT is involved in Bax-induced apoptotic mitochondrial changes. As no mammalian cells lacking ANT were available and Bax could function in yeast cells (Greenhalf et al, 1996;Matsuyama et al, 1998;Shimizu et al, 1999;Gross et al, 2000), we used yeast mutants de®cient for ANT3, one of three ANTs (DANT3), and for all ANTs (DANT1-3). Semi-quantitative PCR analysis (Figure 2a) con®rmed the lack of ANT gene(s) in these mutant yeasts.…”