The gene encoding the yeast mitochondrial outer membrane channel VDAC was subjected to site-directed mutagenesis to change amino acids at 29 positions to residues differing in charge from the wild-type sequence. The mutant genes were then expressed in yeast, and the physiological consequences of single and multiple amino acid changes were assessed after isolation and insertion of mutant channels into phospholipid bilayers. Selectivity changes were observed at 14 sites distributed throughout the length of the molecule. These sites are likely to define the position of the protein walls lining the aqueous pore and hence, the transmembrane segments. These results have been used to develop a model of the open state of the channel in which each polypeptide contributes 12 beta strands and one alpha helix to form the aqueous transmembrane pathway.
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.
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