Figure 3
Yeast and human 14-3-3 proteins are functionally interchangeable. (a) Human 14-3-3β/α can functionally replace the yeast BMH1 and BMH2 encoding the 14-3-3 genes. Freshly saturated cultures of different yeast strains including wild type (WT) as well as the isogenic derivatives that are lacking the yeast 14-3-3 Bmh1 or Bmh2 (Bmh1Δ or Bmh2Δ) genes transformed with different plasmids (Vector, 14-3-3) were serially diluted and aliquots were spotted onto galactose-containing nutrient agar without (no treatment) or with 100 nM rapamycin and the plates were incubated at 30°C. (b–d) Yeast mutants lacking the BMH1 gene are hypersensitive to apoptotic-inducing stress. Yeast mutants lacking the Bmh1Δ and Bmh2Δ genes were treated with cycloheximide and different apoptotic parameters determined. The results are reported as the mean (±S.D.) of two experiments that were performed in triplicate. (b) Viability of the cells was determined after 5 h with cycloheximide using the clonegicity assay. (c) Fluorescence was monitored in DHE-treated cells using a fluorescent cell sorter. (d) Cells were simultaneously challenged with fluorescently labeled annexin V and propidium iodide, and the proportion of cells labeled with either or with both were determined by cell sorter as described.27 (e) Rapamycin-mediated cell death is inhibited by yeast 14-3-3. Spot assays were carried out using freshly saturated cultures of wild-type cells (WT) and mutants lacking 14-3-3 (Bmh1Δ and Bmh2Δ) transformed with control plasmid (Vector) or plasmids overexpressing the yeast 14-3-3 genes (BMH1 and BMH2). The cells were spotted onto galactose-inducible selectable media without (no treatment) or with 150 nM rapamycin (+Rapamycin). *P<0.05 and **P<0.01. (f) Viability was determined, using the vital dye trypan blue, following leucine deprivation in wild-type cells transformed with the empty vector (open diamonds) and the plasmid expressing yeast BMH1 (open triangles). The data are shown as the percentage (%) of cells that does not stain with trypan blue (trypan blue negative). The results are reported as the average of two experiments that were performed in triplicate
