Proximity labeling of trans-cinnamic acid 4-hydroxylase in Petunia inflata petal protoplasts

Proximity-dependent labeling allows untargeted determination of both direct and indirect protein interactions in vivo, and therefore stands as an attractive alternative to targeted binary assays for determining global proteinprotein interaction networks. We used TurboID-based proximity labeling to study protein interaction networks of the core phenylpropanoid and anthocyanin pathways in petunia. To do so, we coupled the endoplasmic reticulum (ER) membrane anchored cytochrome P450 cinnamic acid 4-hydroxylase (C4H, CYP73A412) from Petunia inflata to TurboID and expressed it in protoplasts derived from anthocyanin-rich petunia petals. We identified multiple soluble enzymes from the late anthocyanin pathway among enriched proteins, along with other C4H isoforms, and other ER membrane anchored CYPs. Several of these interactions were subsequently confirmed by bimolecular fluorescence complementation (BiFC).

邻近依赖标记（proximity-dependent labeling）可在体内（in vivo）非靶向地同时测定直接与间接蛋白质相互作用，因此可作为靶向二元测定的优质替代方案，用于解析全局蛋白质-蛋白质相互作用网络。本研究采用基于TurboID的邻近依赖标记技术，探究矮牵牛核心苯丙烷类与花青素通路的蛋白质相互作用网络。具体而言，我们将智利矮牵牛（Petunia inflata）内质网（ER）膜锚定的细胞色素P450肉桂酸4-羟化酶（C4H, CYP73A412）与TurboID融合，并在源自富含花青素的矮牵牛花瓣的原生质体中进行表达。最终在富集蛋白中鉴定出多个晚期花青素通路的可溶性酶、其他C4H同工型以及其他内质网膜锚定的细胞色素P450。后续通过双分子荧光互补（BiFC）验证了其中部分相互作用。