Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress

BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death.BAX INHIBITOR-1 (BI-1) is an endoplasmic reticulum (ER)-based cell death suppressor widely conserved in plants and animals (Xu and Reed, 1998; Kawai et al., 1999). In plants, BI-1 is considered a stress-associated factor, since its expression is stimulated by various stresses (Sanchez et al., 2000; Kawai-Yamada et al., 2001; Matsumura et al., 2003; Watanabe and Lam, 2006; Isbat et al., 2009). Although plants lack the homolog of animal BAX as an inducer of programmed cell death, loss of BI-1 expression results in a severe cell death phenotype under stress conditions, such as fumonisin B1-induced ER stress and disturbance of ion homeostasis (Watanabe and Lam, 2006; Ihara-Ohori et al., 2007). Conversely, plants overexpressing BI-1 exhibit tolerance to cell death induced by various stresses (Kawai-Yamada et al., 2001, 2004; Matsumura et al., 2003; Ihara-Ohori et al., 2007; Watanabe and Lam, 2008; Ishikawa et al., 2010). Moreover, BI-1 overexpression confers not only tolerance to oxidative stress-mediated cell death but also enhanced metabolic acclimation involved in energy and redox balance (Ishikawa et al., 2010). The results of these studies indicate that plant BI-1 is potentially useful for engineering stress-tolerant plants. However, little is known about the mode of action of BI-1 in the cell death regulatory pathway (Ishikawa et al., 2011). While overexpression systems sometimes include artificial or off-site effects, the observation that BI-1 overexpression improves stress tolerance suggests the importance of dissecting plants overexpressing it to further address the molecular basis of BI-1 function and cell death and stress tolerance management.As another approach to understand the molecular function of BI-1, screening of candidates interacting biochemically or functionally with BI-1 has been performed. First, Arabidopsis (Arabidopsis thaliana) BI-1 was confirmed to bind to calmodulin, like barley (Hordeum vulgare) MLO protein, a membrane-bound cell death regulator (Kim et al., 2002; Ihara-Ohori et al., 2007). Since the calmodulin-binding ability of BI-1 and MLO is necessary for their cell death-suppressing activity, Ca²⁺ signaling is critically involved in BI-1- and MLO-mediated cell death regulation (Kim et al., 2002; Kawai-Yamada et al., 2009). More recently, it was also demonstrated that the cell death suppression by BI-1 is mediated, at least in part, through fatty acid hydroxylase (FAH) in a Saccharomyces cerevisiae ectopic expression system (Nagano et al., 2009). In addition, Arabidopsis FAHs (AtFAH1 and AtFAH2) interact with BI-1 via cytochrome b₅ at the ER, resulting in the accumulation of 2-hydroxy fatty acids (2-HFAs) in Arabidopsis plants overexpressing BI-1. 2-HFAs are typical components of the ceramide backbone of sphingolipids (Imai et al., 1995; Pata et al., 2010). Although many functions of plant sphingolipids remain to be elucidated, accumulating evidence clearly indicates that sphingolipids and their metabolism are closely involved in cell death regulation and various stress responses in plants (Ng et al., 2001; Liang et al., 2003; Townley et al., 2005; Chen et al., 2008, 2012; Wang et al., 2008; Saucedo-García et al., 2011; Dutilleul et al., 2012; Kӧnig et al., 2012; Nagano et al., 2012; Mortimer et al., 2013), implying that BI-1 plays a role in cell death regulation through sphingolipid metabolism. Sphingolipids are major components of membrane lipids and are at particularly high concentrations in membrane microdomains, known as lipid rafts in animal cells, which are essential for membrane-mediated signaling and act as a sorting platform for targeted protein traffic (Simons and Toomre, 2000; Staubach and Hanisch, 2011). In mammalian cells, sphingomyelin metabolism in lipid rafts plays a vital role in the initiation of apoptotic cell death (Milhas et al., 2010). Recent studies have demonstrated the presence of raft-like membrane microdomains in plant cells and a role for them in defense responses and targeted protein sorting (Peskan et al., 2000; Fujiwara et al., 2009; Minami et al., 2009; Melser et al., 2010; Markham et al., 2011).This study focused on membrane microdomains in relation to BI-1-mediated sphingolipid metabolism. Our findings indicated that BI-1 alters sphingolipid composition in membrane microdomains, and this is accompanied by dynamic changes in a number of detergent-resistant membrane (DRM) proteins involved in cell death regulation.