Functional association of cell death suppressor, Arabidopsis Bax inhibitor‐1, with fatty acid 2‐hydroxylation through cytochrome b₅

Abstract

SummaryBax 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.

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

“…We have shown that 2-hydroxylation of sphingolipid fatty acids in Arabidopsis thaliana is performed by the enzyme fatty acid 2-hydroxylase (FAH) (Nagano et al, 2009(Nagano et al, , 2012a. Both Arabidopsis FAHs (At-FAH1 and At-FAH2) are localized to the endoplasmic reticulum (ER) membrane, and decreasing their levels affects the response to biotic and abiotic stresses (Nagano et al, 2009(Nagano et al, , 2012aKönig et al, 2012).…”

Bax inhibitor-1: a highly conserved endoplasmic reticulum-resident cell death suppressor

“…Interestingly, AtFAH1 and AtFAH2, the two Arabidopsis homologs of ScFAH1, lack the N-terminal Cb5-like domain and are thus distinct from their yeast and animal counterparts. 56,57 As the two AtFAHs can interact with AtCb5, the biochemical activities of AtBI-1 and AtCb5-AtFAH may be tightly coupled through physical interaction between these proteins. 56 Surprisingly, it was shown that AtBI-1 overexpressing Arabidopsis plants accumulate 2-hydroxylated very long chain fatty acid (VLCFA) species, which are produced by FAHs in yeast and animals.…”

“…56,57 As the two AtFAHs can interact with AtCb5, the biochemical activities of AtBI-1 and AtCb5-AtFAH may be tightly coupled through physical interaction between these proteins. 56 Surprisingly, it was shown that AtBI-1 overexpressing Arabidopsis plants accumulate 2-hydroxylated very long chain fatty acid (VLCFA) species, which are produced by FAHs in yeast and animals. The 2-hydroxylated VLCFAs are known components in the ceramide fatty acyl chains of sphingolipids, 58,59 whereas sphingolipids are believed to be involved in PCD signaling in both plants and animals.…”

“…69,70 This supports the possibility that activation of AtFAH by AtBI-1 contributes to enlargement of the glycosphingolipid pool. 56 The AtBI-1/AtFAH complex may have a role in the formation of membrane microdomains such as 'lipid raft'. These domains are enriched in glycosphingolipids, sterols and certain membrane proteins to form highly ordered and tightly packed structures in lipid bilayers via lipid -lipid, protein -lipid and protein -protein interactions.…”

“…54,55 Cytochrome b 5 and sphingolipid metabolism. Nagano et al 56 isolated cytochrome b 5 (Cb5) as another candidate partner with AtBI-1 by the SuY2H screening method, using an Arabidopsis cDNA expression library. In addition, a yeast mutant lacking ScFAH1, the ER-resident fatty acid hydroxylase containing a Cb5-like domain, failed to rescue Bax lethality of yeast by co-expression of AtBI-1.…”