Arabidopsis Bax Inhibitor-1 inhibits cell death induced by pokeweed antiviral protein in Saccharomyces cerevisiae

Abstract

Apoptosis is an active form of programmed cell death (PCD) that plays critical roles in the development, differentiation and resistance to pathogens in multicellular organisms. Ribosome inactivating proteins (RIPs) are able to induce apoptotic cell death in mammalian cells. In this study, using yeast as a model system, we showed that yeast cells expressing pokeweed antiviral protein (PAP), a single-chain ribosome-inactivating protein, exhibit apoptotic-like features, such as nuclear fragmentation and ROS production. We studied the interaction between PAP and AtBI-1 (Arabidopsis thaliana Bax Inhibitor-1), a plant anti-apoptotic protein, which inhibits Bax induced cell death. Cells expressing PAP and AtBI-1 were able to survive on galactose media compared to PAP alone, indicating a reduction in the cytotoxicity of PAP in yeast. However, PAP was able to depurinate the ribosomes and to inhibit total translation in the presence of AtBI-1. A C-terminally deleted AtBI-1 was able to reduce the cytotoxicity of PAP. Since anti-apoptotic proteins form heterodimers to inhibit the biological activity of their partners, we used a co-immunoprecipitation assay to examine the binding of AtBI-1 to PAP. Both full length and C-terminal deleted AtBI-1 were capable of binding to PAP. These findings indicate that PAP induces cell death in yeast and AtBI-1 inhibits cell death induced by PAP without affecting ribosome depurination and translation inhibition.

Keywords: Arabidopsis thaliana Bax Inhibitor-1; apoptotic-like cell death; pokeweed antiviral protein (PAP); ribosome inactivating proteins (RIPs).

Figure 1. FIGURE 1: Analysis of cell death and nuclear fragmentation in yeast cells expressing PAP, AtBI-1 and AtBI-1∆C.

(A) Cells were stained with Evans Blue or DAPI at 24 h after induction and visualised using Zeiss Axiovert 200 inverted microscope (magnification, X 40) nuclei are shown enlarged 40 times relative to the yeast cells. (B) The percentage of the cell death at different hours after induction were quantified and are represented as the means ± standard deviation (n=3). (C) DAPI stained nuclei at 24 h post-induction were quantified and are represented as the means ± standard deviation (n=3). At least 100 cells were counted per experiment. The results represent three independent experiments. VC - vector control. Columns are statistically different according to ANOVA (P < 0.001) followed by a post-hoc Fisher's Least Significant Difference (LSD) test.

Figure 2. FIGURE 2: ROS generation in cells expressing PAP and AtBI-1.

The amount of H₂O₂ production was quantified using DCDHF-DA. The results are represented as the means ± standard deviation (n=3). VC - vector control. The results represent three independent experiments. Columns are statistically different according to ANOVA (P < 0.001) followed by a post-hoc Fisher's Least Significant Difference (LSD) test.

Figure 3. FIGURE 3: Expression and functional characterization of AtBI-1.

Yeast cells containing AtBI-1 or PAP were grown in SD-U-L/raf overnight then diluted into SD-U-L/galactose for induction at 4 h for PAP and AtBI-1, then serial dilution were spotted on glucose containing plates and incubated for 48 h at 30°C.

Figure 4. FIGURE 4: Ribosome depurination in yeast cells.

Primer extension analysis in yeast cells expressing PAP and AtBI-1 (A) or PAP and AtBI-1∆C (B) using two different end labeled primers, the depurination primer (Dep) was used to measure the extend of depurination and the 25S rRNA primer (25S) was used to measure the amount of 25S rRNA (C) and ratio of Dep/25S (D). The results represent three independent experiments. Columns are statistically different according to ANOVA (P < 0.001) followed by a post-hoc Fisher's Least Significant Difference (LSD) test.

Figure 5. FIGURE 5: Analysis of total translation in yeast cells.

(A) Total translation in yeast cells expressing PAP and AtBI-1. (B) Total translation in yeast cells expressing PAP and AtBI-1∆C. Yeast cells were grown in SD-L-U-Met and 2% glucose overnight then switched to 2% galactose containing media to induce the expression. At time 0, methionine was added to cells growing on galactose which expressed PAP or AtBI-1 and incorporation of methionine was determined at the indicated times. Each point was repeated in duplicate. The results represent three independent experiments. VC - vector control. Columns are statistically different according to ANOVA (P < 0.001) followed by a post-hoc Fisher's Least Significant Difference (LSD) test.

Figure 6. FIGURE 6: Real-time PCR analysis of mRNA levels in yeast cells.

(A) Analysis of PAP mRNA in yeast cells expressing AtBI-1. (B) Analysis of AtBI-1 mRNA in yeast cells expressing PAP. Cells were grown on galactose for the hours indicated. The mRNA levels for the genes were normalized to G6PD mRNA using the ∆∆CT method from Applied Biosystems. The results represent three independent experiments. Columns are statistically different according to ANOVA (P < 0.001) followed by a post-hoc Fisher's Least Significant Difference (LSD) test.

Figure 7. FIGURE 7: Immunoblot analysis.

(A) PAP expression in yeast cells expressing AtBI-1 or AtBI-1ΔC. (B) AtBI-1 or AtBI-1ΔC expression in yeast cells expressing PAP. Yeast transformants were grown in SD-U-L glucose media. The expression of PAP and AtBI-1 was induced for indicated hours, lysed and fractionated into membrane (P18) and cytosolic (S18) components. The amount of 10 mg protein was separated on 15 % SDS-PAGE. Proteins were transferred into nitrocellulose membrane and probed with polyclonal PAP antiserum and monoclonal V5 antibody. The membrane marker Dpm1p and the cytosolic marker Pgk1p were used as controls to show equal amount of loading and lack of cross-contamination.

Figure 8. FIGURE 8: Co-immunoprecipitation assay.

(A) Co-immunoprecipitation of PAP with either AtBI-1 or AtBI-1∆C. Total proteins isolated from yeast cells induced to express PAP, AtBI-1, AtBI-1∆C and vector control (VC) for 6 hours were incubated with V5-antibody and immunoprecipitated with protein A-Sepharose beads. Immunoprecipitated proteins were separated on 15 %SDS-PAGE, transferred to nitrocellulose and probed with affinity purified PAP antibody or with V5-antibody. (B) The total lysate from cells expressing PAP, AtBI-1 and AtBI-1∆C subjected to SDS PAGE/immunoblot analysis using V5 and PAP-antibody to show the level of expression of both proteins.