Proximity-dependent biotin identification (BioID) has emerged as a powerful methodology to identify proteins co-localizing with a given bait protein in vivo. The approach has been established in animal cells, plants and yeast but not yet in filamentous fungi. BioID relies on promiscuous biotin ligases fused to bait proteins to covalently label neighboring proteins with biotin. Biotinylated proteins are specifically enriched through biotin affinity capture from denatured cell lysates and subsequently identified and quantified with liquid chromatography-mass spectrometry (LC–MS). In contrast to many other affinity capture approaches for studying protein–protein interactions, BioID does not rely on physical protein–protein binding within native cell lysates. This feature allows the identification of protein proximities of weak or transient and dynamic nature. Here, we demonstrate the application of BioID for the fungal model organism Sordaria macrospora (Sm) using the example of the STRIPAK complex interactor 1 (SCI1) of the well-characterized striatin-interacting phosphatase and kinase (SmSTRIPAK) complex as proof of concept. For the establishment of BioID in S. macrospora, a codon-optimized TurboID biotin ligase was fused to SCI1. Biotin capture of the known SmSTRIPAK components PRO11, SmMOB3, PRO22 and SmPP2Ac1 demonstrates the successful BioID application in S. macrospora. BioID proximity labeling approaches will provide a powerful proteomics tool for fungal biologists.

接近依赖性生物锡识别(BioID) 已成为一种强大的方法，可在体内识别与给定诱饵蛋白共定位的蛋白质。该方法已在动物细胞、植物和酵母中建立，但尚未在丝状真菌中建立。BioID 依赖于与诱饵蛋白融合的混杂生物素连接酶，用生物素共价标记邻近的蛋白质。生物素化蛋白质通过生物素亲和捕获从变性细胞裂解物中特异性富集，随后使用液相色谱-质谱进行鉴定和定量光谱法（LC-MS）。与许多其他用于研究蛋白质-蛋白质相互作用的亲和捕获方法相比，BioID 不依赖于天然细胞裂解物中的物理蛋白质-蛋白质结合。此功能允许识别弱或瞬态和动态性质的蛋白质接近度。在这里，我们演示了 BioID 在真菌模型生物Sordaria macrospora (Sm) 中的应用，以具有良好特征的链霉菌相互作用磷酸酶和k的S TRIPAK复合物相互作用因子1 ( SCI1 )为例inase (SmSTRIPAK) 复合物作为概念证明。为了在S. macrospora中建立 BioID ，将密码子优化的 TurboID 生物素连接酶与 SCI1 融合。已知 SmSTRIPAK 组件 PRO11、SmMOB3、PRO22 和 SmPP2Ac1 的生物素捕获证明了 BioID 在S. macrospora 中的成功应用。BioID 临近标记方法将为真菌生物学家提供强大的蛋白质组学工具。