Arabidopsis Bax inhibitor-1 interacts with enzymes related to very-long-chain fatty acid synthesis
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Bax inhibitor-1 (BI-1) is a widely conserved cell death regulator that confers resistance to environmental stress in plants. Previous studies suggest that Arabidopsis thaliana BI-1 (AtBI-1) modifies sphingolipids by interacting with cytochrome b5 (AtCb5), an electron-transfer protein. To reveal how AtBI-1 regulates sphingolipid synthesis, we screened yeast sphingolipid-deficient mutants and identified yeast ELO2 and ELO3 as novel enzymes that are essential for AtBI-1 function. ELO2 and ELO3 are condensing enzymes that synthesize very-long-chain fatty acids (VLCFAs), major fatty acids in plant sphingolipids. In Arabidopsis, we identified four ELO homologs (AtELO1–AtELO4), localized in the endoplasmic reticulum membrane. Of those AtELOs, AtELO1 and AtELO2 had a characteristic histidine motif and were bound to AtCb5-B. This result suggests that AtBI-1 interacts with AtELO1 and AtELO2 through AtCb5. AtELO2 and AtCb5-B also interact with KCR1, PAS2, and CER10, which are essential for the synthesis of VLCFAs. Therefore, AtELO2 may participate in VLCFA synthesis with AtCb5 in Arabidopsis. In addition, our co-immunoprecipitation/mass spectrometry analysis demonstrated that AtBI-1 forms a complex with AtELO2, KCR1, PAS2, CER10, and AtCb5-D. Furthermore, AtBI-1 contributes to the rapid synthesis of 2-hydroxylated VLCFAs in response to oxidative stress. These results indicate that AtBI-1 regulates VLCFA synthesis by interacting with VLCFA-synthesizing enzymes.
KeywordsBax inhibitor-1 Oxidative stress Sphingolipid Very-long-chain fatty acid
Plasmids and strains used for cell death assays in yeast were kindly provided by Dr. John C. Reed (The Burnham Institute, La Jolla, CA). Plasmids and strains used for the suY2H system were generously provided by Dr. Ralph Panstruga (Max-Planck Institute, Saabruecken, Germany) and Dr. Imre E. Somssich (Max-Planck Institute). Plasmids used for the BiFC assay were kindly provided by Dr. Tsuyoshi Nakagawa (Shimane University, Japan). We appreciate Dr. Noriko Inada (Nara Institute of Science and Technology, Japan) for technical advice of confocal laser microscopy and FRET analysis. This research was supported by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Fellows Grant Numbers 07J03838 and 11J08349 to M. N.; a Grant in-Aid to C. K. for Scientific Research for Plant Graduate Student from Nara Institute Science and Technology; JSPS KAKENHI Grant Numbers 26850232 and 17K15412 to M. N; 26292190 to M. K-Y..
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