Pupylation-Based Proximity-Tagging of FERONIA-Interacting Proteins in Arabidopsis.

IF 6.1 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS Molecular & Cellular Proteomics Pub Date : 2024-08-13 DOI:10.1016/j.mcpro.2024.100828

Zhuoran Lin, Di Liu, Yifan Xu, Mengyang Wang, YongQi Yu, Andrew C Diener, Kun-Hsiang Liu

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Abstract

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.

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拟南芥中与 FERONIA 相互作用的蛋白质的基于稚嫩化的近似标记。

质膜定位的受体激酶 FERONIA（FER）在拟南芥整个生命周期的各种生物过程中发挥着关键作用。要揭示支撑这些过程的 FER 分子联系，首先要确定与 FER 相互作用的蛋白质。我们采用基于幼体化的相互作用标记（PUP-IT）来调查与 FER 接近的细胞蛋白，包括膜蛋白难以捕捉的微弱和瞬时相互作用。我们在原生质体、幼苗和花朵中分别重复鉴定出了 581、115 和 736 个特异的 FER 相互作用候选蛋白。我们还确认了 14 个先前表征的 FER 相互作用蛋白。原生质体瞬时基因表达加快了用于 PUP-IT 分析的新基因构建物的测试和候选蛋白的验证。我们验证了五种候选蛋白的近似标记，这些蛋白以前未被鉴定为 FER 相互作用蛋白。PUP-IT 方法可以作为一种有价值的工具，用于调查和验证植物中不同亚细胞区室中感兴趣的目标蛋白质之间的相互作用。

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来源期刊

Molecular & Cellular Proteomics 生物-生化研究方法

CiteScore

11.50

自引率

4.30%

发文量

131

审稿时长

84 days

期刊介绍： The mission of MCP is to foster the development and applications of proteomics in both basic and translational research. MCP will publish manuscripts that report significant new biological or clinical discoveries underpinned by proteomic observations across all kingdoms of life. Manuscripts must define the biological roles played by the proteins investigated or their mechanisms of action. The journal also emphasizes articles that describe innovative new computational methods and technological advancements that will enable future discoveries. Manuscripts describing such approaches do not have to include a solution to a biological problem, but must demonstrate that the technology works as described, is reproducible and is appropriate to uncover yet unknown protein/proteome function or properties using relevant model systems or publicly available data. Scope: -Fundamental studies in biology, including integrative "omics" studies, that provide mechanistic insights -Novel experimental and computational technologies -Proteogenomic data integration and analysis that enable greater understanding of physiology and disease processes -Pathway and network analyses of signaling that focus on the roles of post-translational modifications -Studies of proteome dynamics and quality controls, and their roles in disease -Studies of evolutionary processes effecting proteome dynamics, quality and regulation -Chemical proteomics, including mechanisms of drug action -Proteomics of the immune system and antigen presentation/recognition -Microbiome proteomics, host-microbe and host-pathogen interactions, and their roles in health and disease -Clinical and translational studies of human diseases -Metabolomics to understand functional connections between genes, proteins and phenotypes