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

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.