A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast

The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the mitochondrial membrane potential, and alters the intracellular redox potential. Co-expression of the BI-GST/GPX protein brought the total glutathione levels back to normal and re-established the mitochondrial membrane potential but had no effect on the phospholipid alterations. Moreover, expression of BI-GST/GPX in yeast was found to significantly enhance resistance to H(2)O(2)-induced stress. These results underline the relationship between oxidative stress and Bax-induced death in yeast cells and demonstrate that the yeast-based genetic strategy described here is a powerful tool for the isolation of novel antioxidant and antiapoptotic genes.     

Bax-induced cell death of Arabidopsisis meditated through reactive oxygen-dependent and -independent processes

An Arabidopsisprotoplast system was developed for dissecting plant cell death in individual cells. Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces apoptotic-like cell death in Arabidopsis. Bax accumulation in Arabidopsismesophyll protoplasts expressing murine BaxcDNA from a glucocorticoid-inducible promoter results in cytological characteristics of apoptosis, namely DNA fragmentation, increased vacuolation, and loss of plasma membrane integrity. In vivotargeting analysis monitored using jellyfish green fluorescent protein (GFP) reporter indicated full-length Bax was localized to the mitochondria, as it does in animal cells. Deletion of the carboxyl-terminal transmembrane domain of Bax completely abolished targeting to mitochondria. Bax expression was followed by reactive oxygen species (ROS) accumulation. Treatment of protoplasts with the antioxidant N-acetyl- -cysteine (NAC) during induction of Bax expression strongly suppressed Bax-mediated ROS production and the cell death phenotype. However, some population of the ROS depleted cells still induced cell death, indicating that there is a process that Bax-mediated plant cell death is independent of ROS accumulation. Accordingly, suppression of Bax-mediated plant cell death also takes place in two different processes. Over-expression of a key redox-regulator, Arabidopsisnucleoside diphosphate kinase 2 (AtNDPK2) down-regulated ROS accumulation and suppressed Bax-mediated cell death and transient expression of ArabidopsisBax inhibitor-1 (AtBI-1) substantially suppressed Bax-induced cell death without altering cellular ROS level. Taken together, our results collectively suggest that the Bax-mediated cell death and its suppression in plants is mediated by ROS-dependent and -independent processes.     

Induction of mammalian cell death by a plant Bax inhibitor

Arabidopsis thaliana AtBI-1 is an orthologue of mammalian Bax inhibitor-1 capable of suppressing Bax-induced cell death in yeast as well as mammalian cells. Here we investigated whether or not AtBI-1 suppresses Bax-induced cell death using human fibrosarcoma HT1080 cells. Surprisingly, AtBI-1 did not block Bax-induced cell death, but it triggered apoptotic cell death in mammalian cells. The proapoptotic effect of AtBI-1 was blocked by the X-linked caspase inhibitor XIAP, suggesting that the cell death caused by AtBI-1 is similar to that caused by Bax.     

Role of oxidative phosphorylation in Bax toxicity

The Bcl-2-related protein Bax is toxic when expressed either in yeast or in mammalian cells. Although the mechanism of this toxicity is unknown, it appears to be similar in both cell types and dependent on the localization of Bax to the outer mitochondrial membrane. To investigate the role of mitochondrial respiration in Bax-mediated toxicity, a series of yeast mutant strains was created, each carrying a disruption in either a component of the mitochondrial electron transport chain, a component of the mitochondrial ATP synthesis machinery, or a protein involved in mitochondrial adenine nucleotide exchange. Bax toxicity was reduced in strains lacking the ability to perform oxidative phosphorylation. In contrast, a respiratory-competent strain that lacked the outer mitochondrial membrane Por1 protein showed increased sensitivity to Bax expression. Deficiencies in other mitochondrial proteins did not affect Bax toxicity as long as the ability to perform oxidative phosphorylation was maintained. Characterization of Bax-induced toxicity in wild-type yeast demonstrated a growth inhibition that preceded cell death. This growth inhibition was associated with a decreased ability to carry out oxidative phosphorylation following Bax induction. Furthermore, cells recovered following Bax-induced growth arrest were enriched for a petite phenotype and were no longer able to grow on a nonfermentable carbon source. These results suggest that Bax expression leads to an impairment of mitochondrial respiration, inducing toxicity in cells dependent on oxidative phosphorylation for survival. Furthermore, Bax toxicity is enhanced in yeast deficient in the ability to exchange metabolites across the outer mitochondrial membrane.     

Mammalian prion protein suppresses Bax-induced cell death in yeast

Several lines of evidence suggest that PrP(C), the non-infectious form of the prion protein, may function to protect neurons and other cells from stress or toxicity. In this paper, we report on the use of the yeast Saccharomyces cerevisiae as a model system to assay the cytoprotective activity of PrP(C). The mammalian pro-apoptotic protein, Bax, confers a lethal phenotype when expressed in yeast. Since overexpression of PrP(C) has been found to prevent Bax-mediated cell death in cultured human neurons, we explored whether PrP could also suppress Bax-induced cell death in yeast. We utilized a form of mouse PrP containing a modified signal peptide that we had previously shown is efficiently targeted to the secretory pathway in yeast. We found that this PrP potently suppressed the death of yeast cells expressing mammalian Bax under control of a galactose-inducible promoter. In contrast, cytosolic PrP-(23-231) failed to rescue growth of Bax-expressing yeast, indicating that protective activity requires targeting of PrP to the secretory pathway. Deletion of the octapeptide repeat region did not affect the rescuing activity of PrP, but deletion of a charged region encompassing residues 23-31 partially eliminated activity. We also tested several PrP mutants associated with human familial prion diseases and found that only a mutant containing nine extra octapeptide repeats failed to suppress Bax-induced cell death. These findings establish a simple and genetically tractable system for assaying a putative biological activity of PrP(C).     

Bax-induced cell death in yeast depends on mitochondrial lipid oxidation.

The oxidant function of pro-apoptotic protein Bax was investigated through heterologous expression in yeast. Direct measurements of fatty acid content show that Bax-expression induces oxidation of mitochondrial lipids. This effect is prevented by the coexpression of Bcl-xL. The oxidation actually could be followed on isolated mitochondria as respiration-induced peroxidation of polyunsaturated cis-parinaric acid and on whole cells as the increase in the amount of thiobarbituric acid-reactive products. Treatments that increase the unsaturation ratio of lipids, making them more sensitive to oxidation, increase kinetics of Bax-induced death. Conversely, inhibitors of lipid oxidation and treatments that decrease the unsaturation ratio of fatty acids decrease kinetics of Bax-induced death. Taken together, these results show that Bax-induced mitochondrial lipid oxidation is relevant to Bax-induced cell death. Conversely, lipid oxidation is poorly related to the massive Bax-induced superoxide and hydrogen peroxide accumulation, which occurs at the same time, as chemical or enzymatic scavenging of ROS does not prevent lipid oxidation nor has any effects on kinetics of Bax-induced cell death. Whatever the origin of mitochondrial lipid oxidation, these data show that it represents a major step in the cascade of events leading to Bax-induced cell death. These results are discussed in the light of the role of lipid oxidation both in mammalian apoptosis and in other forms of cell death in other organisms.     

The Bax lnhibitor-1 needs a functional electron transport chain for cell death suppression

Bax inhibitor-1 (BI-1) is an evolutionarily conserved cell death suppresser in animals, yeast, and plants. In this study, yeast strains carrying single-gene deletions were screened for factors related to cell death suppression by Arabidopsis BI-1 (AtBI-1). Our screen identified mutants that failed to survive Bax-induced lethality even with AtBI-1 coexpression (Bax suppressor). The Deltacox16 strain was isolated as a BI-1-inactive mutant; it was disrupted in a component of the mitochondrial cytochrome c oxidase. Other mutants defective in mitochondrial electron transport showed a similar phenotype. ATP levels were markedly decreased in all these mutants, suggesting that BI-1 requires normal electron transport activity to suppress cell death in yeast.     

Bax-induced cell death in Pichia pastoris

Murine Bax was expressed in the methylotrophic yeast, Pichia pastoris, using the alcohol oxidase 1 (AOX1) or alcohol oxidase 2 (AOX2) promoter and the AOX1 terminator. Upon induction in methanol medium, transformants containing BAX cDNA under control of the strong AOX1 promoter showed complete growth inhibition and extensive cell death. Except for chromatin condensation, morphological changes typical of apoptosis in mammalian cells could not be observed, indicating that the cell death machinery in P. pastoris is marked different from the endogenous cell death program of higher eukaryotes. Staining of Bax-induced cells with propidium iodide indicated that cell death was not correlated with necrosis. Electron microscopic examination revealed no striking differences in cell morphology, but showed few cells with an enlarged vacuole containing spherical bodies, which suggests autophagic cell death.     

Mammalian Bax initiates plant cell death through organelle destruction

Mammalian Bax is known to cause cell death when expressed in plants. We examined transgenic plants expressing both Bax and organelle-targeted green fluorescent protein to determine the cellular changes that occur during Bax-induced cell death. The mitochondria changed morphologically from being bacilli-shaped to being round, eventually becoming swollen. Mitochondria streaming also stopped. The chloroplasts lost membrane function and their contents leaked out, followed by the disruption of the vacuole. Light was not essential for Bax-induced ion leakage or organelle disruption. These results indicate that Bax induces temporal and spatial cell death events at the organelle level in the plant. A heterologous system, using Bax, would therefor be available to investigate cell death, which is commonly conserved in animals and plantsElectronic Supplementary Material Supplementary material is available for this article at     

Cell death suppressor Arabidopsis bax inhibitor-1 is associated with calmodulin binding and ion homeostasis

Cell death suppressor Bax inhibitor-1 (BI-1), an endoplasmic reticulum membrane protein, exists in a wide range of organisms. The split-ubiquitin system, overlay assay, and bimolecular fluorescence complementation analysis demonstrated that Arabidopsis (Arabidopsis thaliana) BI-1 (AtBI-1) interacted with calmodulin in yeast (Saccharomyces cerevisiae) and in plant cells. Furthermore, AtBI-1 failed to rescue yeast mutants lacking Ca2+ ATPase (Pmr1 or Spf1) from Bax-induced cell death. Pmr1 and Spf1, p-type ATPases localized at the inner membrane, are believed to be involved in transmembrane movement of calcium ions in yeast. Thus, the presence of intact Ca2+ ATPases was essential for AtBI-1-mediated cell death suppression in yeast. To investigate the effect of AtBI-1 on calcium homeostasis, we evaluated sensitivity against cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase in AtBI-1-overexpressing or knock-down transgenic Arabidopsis plants. These plants demonstrated altered CPA or ion stress sensitivity. Furthermore, AtBI-1-overexpressing cells demonstrated an attenuated rise in cytosolic calcium following CPA or H2O2 treatment, suggesting that AtBI-1 affects ion homeostasis in plant cell death regulation.     

Vesicle-associated membrane protein of Arabidopsis suppresses Bax-induced apoptosis in yeast downstream of oxidative burst

Programmed cell death (PCD) in many systems is controlled by relative amounts of the apoptosis-regulating proteins Bax and Bcl-2 through homo- or heterodimerization. Here we show that Bax-induced PCD of yeast was suppressed by transformation with a vesicle-associated membrane protein from Arabidopsis (AtVAMP), which was isolated by screening a cDNA expression library against sugar-induced cell death in yeast. AtVAMP expression blocked Bax-induced PCD downstream of oxidative burst. AtVAMP also prevented H(2)O(2)-induced apoptosis in yeast and in Arabidopsis cells. Reduced oxidation of lipids and plasma membrane proteins was detected in the AtVAMP-transformed yeast, suggesting improved membrane repair. Inhibition of intracellular vesicle trafficking by brefeldin A induced apoptosis from a sublethal concentration of H(2)O(2). No protection occurred by overexpression of the yeast homolog SCN2. However, efficient suppression of yeast PCD occurred by expression of a chimeric gene, composed of the conserved domains from yeast, fused to the variable N-terminal domain from Arabidopsis, resulting in exchange of the proline-rich N-terminal domain of SCN2 with a proline-poor Arabidopsis sequence. Our results suggest that intracellular vesicle traffic can regulate execution of apoptosis by affecting the rate of membrane recycling and that the proline-rich N-terminal domain of VAMP inhibited this process.     

Identification of cytochrome c oxidase subunit 6A1 as a suppressor of Bax-induced cell death by yeast-based functional screening

Human cytochrome c oxidase subunit VIa polypeptide 1 (COX6A1) was identified as a novel suppressor of Bcl-2-associated X protein (Bax)-mediated cell death using yeast-based functional screening of a mammalian cDNA library. The overexpression of COX6A1 significantly suppressed Bax- and N-(4-hydroxyphenyl)retinamide (4-HPR)-induced apoptosis in yeast and human glioblastoma-derived U373MG cells, respectively. The generation of reactive oxygen species (ROS) in response to Bax or 4-HPR was inhibited in yeast and U373MG cells that expressed COX6A1, indicating that COX6A1 exerts a protective effect against ROS-induced cell damage. 4-HPR-induced mitochondrial translocation of Bax, release of mitochondrial cytochrome c, and activation of caspase-3 were markedly attenuated in U373MG cells that stably expressed COX6A1. Our results demonstrate that yeast-based functional screening of human genes for inhibitors of Bax-sensitivity in yeast identified a protein that not only suppresses the toxicity of Bax in yeast, but also has a potential role in protecting mammalian cells from 4-HPR-induced apoptosis.     

A cytochrome c-GFP fusion is not released from mitochondria into the cytoplasm upon expression of Bax in yeast cells

To study Bax-induced release of cytochrome c in vivo, we have expressed a cytochrome c-GFP (green fluorescent protein) fusion in Saccharomyces cerevisiae cells null for the expression of the endogenous cytochrome. We show here that cytochrome c-GFP is efficiently localised to mitochondria and able to function as an electron carrier between complexes III and IV of the respiratory chain. Strikingly, while natural cytochrome c is released into the cytoplasm upon expression of Bax, the cytochrome c-GFP fusion is not. Nevertheless, cells co-expressing Bax and the cytochrome c-GFP fusion die, indicating that mitochondrial release of cytochrome c is not essential for cell death to occur in yeast. The failure to release cytochrome c-GFP is presumed to arise from increased bulk due to the GFP moiety. We propose that in intact yeast cells, Bax-induced release of cytochrome c into the cytoplasm occurs through a selective pore and not as a consequence of the non-specific breakage of the mitochondrial outer membrane.     

Prion protein prevents Bax-mediated cell death in the absence of other Bcl-2 family members in Saccharomyces cerevisiae

Although there is no consensus regarding the normal function of the prion protein, increasing evidence points towards a role in cellular protection against cell death. We have previously shown that prion protein is a potent inhibitor of Bax-induced apoptosis in human primary neurons and in the breast carcinoma MCF-7 cells. Here, we used the yeast Saccharomyces cerevisiae to investigate if the neuroprotective function of prion protein requires other members of the Bcl-2 family given that S. cerevisiae lacks Bcl-2 genes but undergoes a mitochondrial-dependent apoptotic cell death upon exogenous expression of Bax protein. We show that Bax induces cell death and growth inhibition in S. cerevisiae. Prion protein prevents Bax-mediated cell death. Prion protein overcomes Bax-mediated growth arrest in S phase but cannot overcome population growth inhibition because the cells then accumulate in G(2)/M phase. We conclude that prion protein does not require other Bcl-2 family proteins to protect against Bax-mediated cell death.     

The Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP) can function as a dominant suppressor of Bax-induced cell death of yeast.

We identified genes based on screening of an Arabidopsis cDNA library for functional suppressors of mouse Bax-induced cell death of yeast cells. Interestingly, the cDNA encoding AtEBP, known as Arabidopsis thaliana ethylene-responsive element binding protein, was isolated numerous times in the functional screen (82% of all suppressors). Full-length AtEBP and its localization to the nucleus were essential for the suppression of Bax-induced cell death. Morphological abnormality of intracellular network that is a hallmark of Bax-induced cell death was attenuated by expression of AtEBP.     

Tomato phospholipid hydroperoxide glutathione peroxidase inhibits cell death induced by Bax and oxidative stresses in yeast and plants

Using a conditional life or death screen in yeast, we have isolated a tomato (Lycopersicon esculentum) gene encoding a phospholipid hydroperoxide glutathione peroxidase (LePHGPx). The protein displayed reduced glutathione-dependent phospholipid hydroperoxide peroxidase activity, but differs from counterpart mammalian enzymes that instead contain an active seleno-Cys. LePHGPx functioned as a cytoprotector in yeast (Saccharomyces cerevisiae), preventing Bax, hydrogen peroxide, and heat stress induced cell death, while also delaying yeast senescence. When tobacco (Nicotiana tabacum) leaves were exposed to lethal levels of salt and heat stress, features associated with mammalian apoptosis were observed. Importantly, transient expression of LePHGPx protected tobacco leaves from salt and heat stress and suppressed the apoptotic-like features. As has been reported, conditional expression of Bax was lethal in tobacco, resulting in tissue collapse and membrane permeability to Evans blue. When LePHGPx was coexpressed with Bax, little cell death and no vital staining were observed. Moreover, stable expression of LePHGPx in tobacco conferred protection against the fungal phytopathogen Botrytis cinerea. Taken together, our data indicated that LePHGPx can protect plant tissue from a variety of stresses. Moreover, functional screens in yeast are a viable tool for the identification of plant genes that regulate cell death.     

The mitochondrial fission regulator DRP3B does not regulate cell death in plants

BACKGROUND AND AIMS: Recent reports have described dramatic alterations in mitochondrial morphology during metazoan apoptosis. A dynamin-related protein (DRP) associated with mitochondrial outer membrane fission is known to be involved in the regulation of apoptosis. This study analysed the relationship between mitochondrial fission and regulation of plant cell death. METHODS: Transgenic plants were generated possessing Arabidopsis DRP3B (K56A), the dominant-negative form of Arabidopsis DRP, mitochondrial-targeted green fluorescent protein and mouse Bax. KEY RESULTS: Arabidopsis plants over-expressing DRP3B (K56A) exhibited long tubular mitochondria. In these plants, mitochondria appeared as a string-of-beads during cell death. This indicates that DRP3B (K56A) prevented mitochondrial fission during plant cell death. However, in contrast to results for mammalian cells and yeast, Bax-induced cell death was not inhibited in DRP3B (K56A)-expressing plant cells. Similarly, hydrogen peroxide-, menadione-, darkness- and salicylic acid-induced cell death was not inhibited by DRP3B (K56A) expression. CONCLUSIONS: These results indicate that the systems controlling cell death in animals and plants are not common in terms of mitochondrial fission.