State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest Agriculture & Forestry University, Yangling, Shaanxi, China.
Department of Molecular Biology, Centre for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA; Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Mol Cell Proteomics. 2024 Nov;23(11):100828. doi: 10.1016/j.mcpro.2024.100828. Epub 2024 Aug 13.
The plasma membrane-localized receptor kinase FERONIA (FER) plays critical roles in a remarkable variety of biological processes throughout the life cycle of Arabidopsis thaliana. Revealing the molecular connections of FER that underlie these processes starts with identifying the proteins that interact with FER. We applied pupylation-based interaction tagging (PUP-IT) to survey cellular proteins in proximity to FER, encompassing weak and transient interactions that can be difficult to capture for membrane proteins. We reproducibly identified 581, 115, and 736 specific FER-interacting protein candidates in protoplasts, seedlings, and flowers, respectively. We also confirmed 14 previously characterized FER-interacting proteins. Protoplast transient gene expression expedited the testing of new gene constructs for PUP-IT analyses and the validation of candidate proteins. We verified the proximity labeling of five selected candidates that were not previously characterized as FER-interacting proteins. The PUP-IT method could be a valuable tool to survey and validate protein-protein interactions for targets of interest in diverse subcellular compartments in plants.
质膜定位受体激酶 FERONIA（FER）在拟南芥整个生命周期的许多生物学过程中发挥着关键作用。揭示 FER 作为这些过程基础的分子联系，首先要确定与 FER 相互作用的蛋白质。我们应用基于泛素化的相互作用标记（PUP-IT）来检测与 FER 接近的细胞蛋白，包括难以捕获的膜蛋白的弱和瞬时相互作用。我们分别在原生质体、幼苗和花朵中可重复地鉴定了 581、115 和 736 个特定的 FER 相互作用蛋白候选物。我们还证实了 14 种先前表征的 FER 相互作用蛋白。原生质体瞬时基因表达加快了对新基因构建体的测试，以进行 PUP-IT 分析和候选蛋白的验证。我们验证了五个先前未被表征为 FER 相互作用蛋白的选定候选物的接近标记。PUP-IT 方法可能是一种有价值的工具，可用于在植物的不同亚细胞区室中检测和验证感兴趣的靶标蛋白-蛋白相互作用。
