RESUMO
Bax inhibitor-1 (BI-1) is a cell death suppressor protein conserved across a variety of organisms. The Arabidopsis atbi1-1 plant is a mutant in which the C-terminal 6 amino acids of the expressed BI-1 protein have been replaced by T-DNA insertion. This mutant BI-1 protein (AtBI-CM) produced in Escherichia coli can no longer bind to calmodulin. A promoter-reporter assay demonstrated compartmentalized expression of BI-1 during hypersensitive response, introduced by the inoculation of Pseudomonas syringae possessing the avrRTP2 gene, Pst(avrRPT2). In addition, both BI-1 knockdown plants and atbi1-1 showed increased sensitivity to Pst(avrRPT2)-induced cell death. The results indicated that the loss of calmodulin binding reduces the cell death suppressor activity of BI-1 in planta.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/microbiologia , Arabidopsis/fisiologia , Calmodulina/metabolismo , Morte Celular/fisiologia , Proteínas de Membrana/metabolismo , Pseudomonas syringae/fisiologia , Sequência de Aminoácidos , Arabidopsis/anatomia & histologia , Proteínas de Arabidopsis/genética , Calmodulina/classificação , Calmodulina/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Filogenia , Plantas Geneticamente Modificadas , Ligação ProteicaRESUMO
Bax inhibitor-1 (BI-1) is a widely conserved cytoprotective protein localized in the endoplasmic reticulum (ER) membrane. We identified Arabidopsis cytochrome b(5) (AtCb5) as an interactor of Arabidopsis BI-1 (AtBI-1) by screening the Arabidopsis cDNA library with the split-ubiquitin yeast two-hybrid (suY2H) system. Cb5 is an electron transfer protein localized mainly in the ER membrane. In addition, a bimolecular fluorescence complementation (BiFC) assay and fluorescence resonance energy transfer (FRET) analysis confirmed that AtBI-1 interacted with AtCb5 in plants. On the other hand, we found that the AtBI-1-mediated suppression of cell death in yeast requires Saccharomyces cerevisiae fatty acid hydroxylase 1 (ScFAH1), which had a Cb5-like domain at the N terminus and interacted with AtBI-1. ScFAH1 is a sphingolipid fatty acid 2-hydroxylase localized in the ER membrane. In contrast, AtFAH1 and AtFAH2, which are functional ScFAH1 homologues in Arabidopsis, had no Cb5-like domain, and instead interacted with AtCb5 in plants. These results suggest that AtBI-1 interacts with AtFAHs via AtCb5 in plant cells. Furthermore, the overexpression of AtBI-1 increased the level of 2-hydroxy fatty acids in Arabidopsis, indicating that AtBI-1 is involved in fatty acid 2-hydroxylation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocromos b5/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Arabidopsis/classificação , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Morte Celular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos b5/classificação , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Teste de Complementação Genética , Hidroxilação , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Dados de Sequência Molecular , Filogenia , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/genética , Morte Celular , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
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.
