FIG. 1.
BAX expression in yeast results in growth arrest that is inhibited by BCL-XL. (A) The top panel shows expression of BAX and BCL-XL in yeast. Yeast cells harboring ADH-BCL-XL (left) or GAL-BAX (right) expression vector were washed in water and resuspended in SG medium. Whole-cell lysates were prepared at the indicated time points. BAX and BCL-XL were immunoprecipitated using anti-mBAX 4D2 MAb or anti-mBCL-XL 13.6 polyclonal Ab, respectively, and then size fractionated on SDS–16% polyacrylamide gels. Western blots were developed with anti-mBAX Ab 651 or anti-mBCL-XL Ab 13.6. One asterisk denotes a potentially modified form of BAX, and two asterisks denote the ∼p18 cleaved form of BAX. The bottom panel shows that cells carrying BAX alone or BAX together with BCL-XL express similar levels of BAX. Yeast cells harboring GAL-BAX and ADH–BCL-XL (left lane) or only GAL-BAX (right lane) expression vectors were washed in water and resuspended in SG medium. Whole-cell lysates were prepared 24 h after the transfer to SG medium. BAX was immunoprecipitated using anti-mBAX 4D2 MAb and then size fractionated on SDS–16% polyacrylamide gels. Western blots were developed with anti-mBAX Ab 651. One asterisk denotes a potentially modified form of BAX, and two asterisks denote the ∼p18 cleaved form of BAX. Of note, coexpression of BCL-XL eliminates the appearance of the modified and the cleavage product of BAX. (B) Cells harboring GAL-BAX fail to grow on galactose-based plates. YM1372 yeast cells were cotransformed with either the empty vectors (GAL Vector, ADH Vector), GAL vector and ADH-BCL-XL (GAL Vector, ADH BCL-XL), GAL-BAX and ADH vector (GAL BAX, ADH Vector), or GAL-BAX and ADH-BCL-XL (GAL BAX, ADH BCL-XL). Each transformant was grown on glucose-based and galactose-based plates and incubated at 30°C for 2 days.