Dataset Information

TurboID-mediated proximity labeling for screening interacting proteins of FIP37 in Arabidopsis

ABSTRACT: Proximity labeling (PL) was recently developed to detect protein–protein interactions (PPIs) and members of subcellular multiprotein structures in living cells. Proximity labeling is conducted by fusing an engineered enzyme with catalytic activity, such as biotin ligase, to a protein of interest (bait protein) to biotinylate adjacent proteins. The biotinylated protein can be purified by strep-tavidin beads, and identified by mass spectrometry (MS). TurboID is an engineered biotin ligase with high catalytic efficiency, which is used for proximity labeling. Although TurboID-based proximity labeling technology has been successfully established in mammals, its application in plant systems is limited. Here, we report the usage of TurboID for proximity labeling of FIP37, a core member of m6A methyltransferase complex, to identify FIP37 interacting proteins in Ara-bidopsis thaliana. By analyzing the MS data, we found 214 proteins biotinylated by GFP-TurboID-FIP37 fusion, including five components of m6A methyltransferase complex that have been previously confirmed. Therefore, the identified proteins may include potential proteins directly involved in the m6A pathway or functionally related to m6A-coupled mRNA processing due to spatial proximity. Moreover, we demonstrated the feasibility of proximity labeling tech-nology in plant epitranscriptomics studies, thereby expanding the application of this technology to more subjects of plant research.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Whole Body

SUBMITTER: li xiao fang

LAB HEAD: Chuanlin Shi

PROVIDER: PXD043041 | Pride | 2024-01-26

REPOSITORIES: Pride

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Publications

TurboID-mediated proximity labeling for screening interacting proteins of FIP37 in <i>Arabidopsis</i>.

Li Xiaofang X Wei Yanping Y Fei Qili Q Fu Guilin G Gan Yu Y Shi Chuanlin C

Plant direct 20231218 12

Proximity labeling was recently developed to detect protein-protein interactions and members of subcellular multiprotein structures in living cells. Proximity labeling is conducted by fusing an engineered enzyme with catalytic activity, such as biotin ligase, to a protein of interest (bait protein) to biotinylate adjacent proteins. The biotinylated protein can be purified by streptavidin beads, and identified by mass spectrometry (MS). TurboID is an engineered biotin ligase with high catalytic e ...[more]

PMID: 38111714

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Project description:Identifying specific protein interactors and spatially or temporally restricted local proteomes contributes significantly to our understanding of cellular processes in virtually all aspects of life. Obtaining such data is challenging, especially when the protein, cell type or event of interest is rare. In recent years, different proximity labeling techniques have been developed that have greatly improved our ability to tackle these questions. However, while effective in mammalian systems, use in plants has been extremely limited due to technical challenges. Recent technological improvements in the form of two highly active versions of the biotin ligase BirA* (TurboID and miniTurboID), prompted us to test this new system on two challenging but widely asked questions in plants: what are interaction partners of low-abundant proteins and what are organellar proteomes in rare and transient cell types. To address the first question, we used the transcription factor FAMA as a test case. FAMA is a master regulator of guard cell development and promotes terminal differentiation of the guard cell precursor by both activating and repressing hundreds of genes. FAMA-expressing young guard cells are rare and restricted to the epidermis of developing aerial tissues, which makes them a good model system to test TurboID applicability under material-limiting conditions. For this experiment, young Arabidopsis seedlings expressing FAMA-TurboID were treated with biotin to label FAMA complexes. Col-0 wild type and seedlings expressing nuclear TurboID under the FAMA promoter were used as controls for unspecific binding of proteins to the beads and stochastic labeling of nuclear proteins, respectively. Biotinylated proteins were isolated by affinity purification with streptavidin-coupled beads and identified by LC-MS/MS. Analysis of proteins labeled by FAMA-TurboID fusions revealed known interactors of this late stomatal lineage specific transcription factor, as well as novel proteins that could explain its dual function as an activator and repressor. Comparison with proteins obtained from classical co-immunoprecipitation approaches with FAMA showed that proximity labeling is superior for identification of meaningful interaction partners of low-abundant proteins.

Dataset Information

TurboID-mediated proximity labeling for screening interacting proteins of FIP37 in Arabidopsis

Publications

TurboID-mediated proximity labeling for screening interacting proteins of FIP37 in <i>Arabidopsis</i>.

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