Waki Toshiyuki, Yoo DongChan, Fujino Naoto, Mameda Ryo, Denessiouk Konstantin, Yamashita Satoshi, Motohashi Reiko, Akashi Tomoyoshi, Aoki Toshio, Ayabe Shin-ichi, Takahashi Seiji, Nakayama Toru
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan.
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan; Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland.
Biochem Biophys Res Commun. 2016 Jan 15;469(3):546-51. doi: 10.1016/j.bbrc.2015.12.038. Epub 2015 Dec 13.
Metabolic enzymes, including those involved in flavonoid biosynthesis, are proposed to form weakly bound, ordered protein complexes, called "metabolons". Some hypothetical models of flavonoid biosynthetic metabolons have been proposed, in which metabolic enzymes are believed to anchor to the cytoplasmic surface of the endoplasmic reticulum (ER) via ER-bound cytochrome P450 isozymes (P450s). However, no convincing evidence for the interaction of flavonoid biosynthetic enzymes with P450s has been reported previously. Here, we analyzed binary protein-protein interactions of 2-hydroxyisoflavanone synthase 1 (GmIFS1), a P450 (CYP93C), with cytoplasmic enzymes involved in isoflavone biosynthesis in soybean. We identified binary interactions between GmIFS1 and chalcone synthase 1 (GmCHS1) and between GmIFS1 and chalcone isomerases (GmCHIs) by using a split-ubiquitin membrane yeast two-hybrid system. These binary interactions were confirmed in planta by means of bimolecular fluorescence complementation (BiFC) using tobacco leaf cells. In these BiFC analyses, fluorescence signals that arose from the interaction of these cytoplasmic enzymes with GmIFS1 generated sharp, network-like intracellular patterns, which was very similar to the ER-localized fluorescence patterns of GmIFS1 labeled with a fluorescent protein. These observations provide strong evidence that, in planta, interaction of GmCHS1 and GmCHIs with GmIFS1 takes place on ER on which GmIFS1 is located, and also provide important clues to understand how enzymes and proteins form metabolons to establish efficient metabolic flux of (iso)flavonoid biosynthesis.
包括参与类黄酮生物合成的酶在内的代谢酶，被认为会形成弱结合的有序蛋白质复合物，即所谓的“代谢体”。已经提出了一些类黄酮生物合成代谢体的假设模型，其中代谢酶被认为通过内质网（ER）结合的细胞色素P450同工酶（P450s）锚定在内质网的细胞质表面。然而，此前尚未有关于类黄酮生物合成酶与P450s相互作用的确凿证据报道。在此，我们分析了大豆中一种P450（CYP93C），即2-羟基异黄酮合酶1（GmIFS1）与参与异黄酮生物合成的细胞质酶之间的二元蛋白质-蛋白质相互作用。我们使用分裂泛素膜酵母双杂交系统鉴定了GmIFS1与查尔酮合酶1（GmCHS1）以及GmIFS1与查尔酮异构酶（GmCHIs）之间的二元相互作用。这些二元相互作用通过使用烟草叶细胞的双分子荧光互补（BiFC）在植物体内得到了证实。在这些BiFC分析中，这些细胞质酶与GmIFS1相互作用产生的荧光信号形成了尖锐的、网络状的细胞内模式，这与用荧光蛋白标记的GmIFS1的内质网定位荧光模式非常相似。这些观察结果提供了强有力的证据，表明在植物体内，GmCHS1和GmCHIs与GmIFS1的相互作用发生在GmIFS1所在的内质网上，也为理解酶和蛋白质如何形成代谢体以建立（异）黄酮生物合成的高效代谢通量提供了重要线索。
