Evolutionarily conserved cytoprotection provided by Bax Inhibitor-1 homologs from animals, plants, and yeast

Abstract

Programmed cell death (PCD) plays important roles in the development and physiology of both animals and plants, but it is unclear whether similar mechanisms are employed. Bax Inhibitor-1 (BI-1) is an intracellular multi-membrane-spanning protein and cell death inhibitor, originally identified by a function-based screen for mammalian cDNAs capable of suppressing cell death in yeast engineered to ectopically express the pro-apoptotic protein Bax. Using this yeast assay, we screened expression libraries for cDNAs from the plant, Lycopersicon esculentum (tomato), and the invertebrate animal Drosophila melanogaster (fruit fly), identifying close homologs of BI-1 as Bax-suppressors. We studied the fly and tomato homologs of BI-1, as well as BI-1 homologs identified in Arabidopsis thaliana, Oryza sativa (rice), and Saccharomyces cerevisiae (budding yeast). All eukaryotic homologs of BI-1 blocked Bax-induced cell death when expressed in yeast. Eukaryotic BI-1 homologs also partially rescued yeast from cell death induced by oxidative stress (H₂O₂) and heat shock. Deletion of a C-terminal domain from BI-1 homologs abrogated their cytoprotective function in yeast, demonstrating conserved structure–function relations among these proteins. Expression of tomato BI-1 by agroinfiltration of intact plant leaves provided protection from damage induced by heat-shock and cold-shock stress. Altogether, these findings indicate that BI-1 homologs exist in multiple eukaryotic species, providing cytoprotection against diverse stimuli, thus implying that BI-1 regulates evolutionary conserved mechanisms of stress resistance that are germane to both plants and animals.

Introduction

Programmed cell death (PCD) plays critical roles in a wide variety of normal physiological processes in multicellular animal species. The genes that control programmed cell death in animals are conserved across wide evolutionary distances, defining a core set of biochemical reactions that are regulated in diverse ways by inputs from myriad upstream pathways (Metzstein et al., 1998). These genes encode either anti-apoptotic and pro-apoptotic proteins, which do battle with each other in making cell life–death decisions.

Programmed cell death (PCD) plays normal physiological roles in a variety of processes in plants, including (a) deletion of cells with temporary functions such as the aleurone cells in seeds and the suspensor cells in embryos; (b) removal of unwanted cells, such as the root cap cells found in the tips of elongating plant roots and the stamen primoridia cells in unisexual flowers; (c) deletion of cells during sculpting of the plant body and formation of leaf lobes and perforations; (d) death of cells during plant specialization, such as the death of TE cells which creates channels for water transport in vascular plants; (e) leaf senescence; and (f) responses to plant pathogens Danon et al., 2000, Pennell and Lamb, 1997. Some elements of the same cell suicide mechanisms used in animal cells may be functionally conserved in plants. Though the biochemical mechanisms responsible for cell suicide in plants are largely unknown, a variety of reports suggest similarities to the PCD that occurs in animal species. For example, PCD in plants typically requires new gene expression and thus can be suppressed by cycloheximide and similar inhibitors of protein or RNA synthesis. The morphological characteristics of plant cells undergoing PCD also bear some striking similarities to apoptosis in animals, though the presence of a cell wall around plant cells imposes certain differences. Akin to animal cells, PCD in plants is associated with inter-nucleosomal DNA fragmentation (DNA ladders) and the activation of proteases Del Pozo and Lam, 1998, Richael et al., 2001, Solomon et al., 1999. Moreover, ectopic expression of certain animal anti-apoptosis genes in transgenic plants has been demonstrated to provide protection from crop-pathogens and other insults as a result of cell death suppression Dickman et al., 2001, Mitsuhara et al., 1999. Conversely, expression of animal pro-apoptotic proteins such as Bax in plants can induce cell death mechanisms similar to endogenous programs for cell suicide (Lacomme and Cruz, 1999). However, to date, few endogenous plant genes have been identified that share sequence homology with the apoptosis genes of animal cells.

Bax Inhibitor-1 (BI-1) is an anti-apoptotic protein which is conserved in both animal and plant species. Though transcripts encoding this protein were known from unrelated experiments, the cytoprotective function of BI-1 was discovered in cDNA library screens for human proteins capable of suppressing death of yeast induced by ectopic expression of mammalian Bax protein, a pro-apoptotic member of the Bcl-2 family of apoptosis-regulating proteins Perfettini et al., 2002, Xu and Reed, 1998. When overexpressed in mammalian cells, BI-1 provides protection against apoptosis induced by several types of stimuli, including Bax overexpression, growth factor deprivation, and Ca²⁺ mobilizing agents (Xu and Reed, 1998). Moreover, BI-1 protects certain types of cells against TRAIL, a member of the tumor necrosis factor (TNF) family of cytokines (Burns and El-Deiry, 2001). Antisense experiments in which BI-1 expression was knocked down suggested that the endogenous BI-1 protein can be important for suppressing apoptosis in some types of tumor cell lines (Xu and Reed, 1998). Immunolocalization and subcellular fractionation studies demonstrated that BI-1 is located predominantly in the endoplasmic reticulum (ER) (Xu and Reed, 1998).

How the BI-1 protein functions is unknown. A Kyte–Doolittle plot of protein hydrophobicity revealed six predicted membrane-spanning domains in mammalian BI-1 proteins (Xu and Reed, 1998). Similar proteins have been identified in several plant species, including Oryza sativa (rice), Arabidopsis thaliana (At), Hordeum vulgare (barley), Brassica napus (oilseed rape), and Nicotiana tabacum (tobacco) Bolduc et al., 2003, Huckelhoven et al., 2003, Kawai et al., 1999, Kawai-Yamada et al., 2001, Lam et al., 2001, Sanchez et al., 2000. Moreover, the function of plant homologues of BI-1 appears to be at least partly conserved with its human counterpart, given that BI-1 orthologs of rice and Arabidopsis also rescue in the yeast-based Bax-lethality assay Kawai et al., 1999, Sanchez et al., 2000 and BI-1 homologs of oilseed rape and tobacco rescue against Bax-induced apoptosis in human cells (Bolduc et al., 2003). Importantly, Arabidopsis BI-1 has been shown to protect transgenic plants against cell death induced by ectopic expression of mammalian Bax (Kawai-Yamada et al., 2001), indicating an in vivo role for BI-1 in cytoprotective pathways in planta and suggesting that the biochemical mechanism regulated by BI-1 is evolutionarily conserved. BI-1 overexpression also regulates resistance to fungal pathogens in barley, probably due to its cell death-suppressive effects (Huckelhoven et al., 2003). Conversely, antisense-mediated downregulation of BI-1 in tobacco BY-2 cells results in accelerated cell death upon carbon starvation (Bolduc and Brisson, 2002). Interestingly, endogenous expression of BI-1 is induced during wound-healing responses and upon exposure to certain pathogens in plants Huckelhoven et al., 2001, Sanchez et al., 2000, suggesting that BI-1 may play a role in host-defense mechanisms during times of stress. In this regard, endogenous BI-1 expression is downregulated by treatment of cultured rice cells (O. sativa) with cytotoxic extracts from rice blast fungus (Magnaporthe grisea), while overexpression of At-BI-1 sustains survival (Matsumura et al., 2003). Thus, BI-1 represents the first endogenous gene to be identified that regulates cell death in both plant and animal cells.

In this report, we used a combination of functional cloning to identify additional BI-1 homologs from plant and animal eukaryotic species and performed a comparative analysis of the BI-1 homologs of humans (Homo sapiens), insects (Drosophila), tomato (Lycopersicon), rice (Oryza), mustard (Arabidopsis), and budding yeast (Saccharomyces cerevisiae). Our findings reveal conservation of function of these eukaryotic BI-1 protein, implying an evolutionarily preserved role for BI-1 in cytoprotection.