Biochemical and genetic analysis of the mitochondrial response of yeast to BAX and BCL-X(L).
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 beta subunit or the delta 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 beta 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.
Gross
A
A
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Pilcher
K
K
Blachly-Dyson
E
E
Basso
E
E
Jockel
J
J
Bassik
M C
MC
Korsmeyer
S J
SJ
Forte
M
M
D017209:Apoptosis; D015153:Blotting, Western; D002455:Cell Division; D005434:Flow Cytometry; D005690:Galactose / Q000378:metabolism; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D015003:Yeasts / Q000235:genetics*; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D005947:Glucose; D005690:Galactose