Dataset Information

Application of TurboID-mediated proximity labeling and phosphoproteomics for mapping a GSK3 kinase signaling network in Arabidopsis

ABSTRACT: Transient protein-protein interactions (PPIs), such as those between posttranslational modifying enzymes and their substrates, play key roles in cellular regulation, but are difficult to identify. Here we demonstrate the application of enzyme-catalyzed proximity labeling (PL), using the engineered promiscuous biotin ligase TurboID, as a sensitive method for characterizing PPIs in signaling networks. We show that TurboID fused with the GSK3-like kinase BIN2 or a PP2A phosphatase biotinylate their known substrate, the BZR1 transcription factor, with high specifically and efficiency. We optimized the protocol of biotin labeling and affinity purification in transgenic Arabidopsis expressing a BIN2-TurboID fusion protein. Subsequent quantitative mass spectrometry (MS) analysis identified about three hundred proteins biotinylated by BIN2-TurboID more efficiently than the YFP-TurboID control. These include a significant subset of previously proven BIN2 interactors and a large number of new BIN2 interactors that uncover a broad BIN2 signaling network. Our study illustrates that PL-MS using TurboID is a powerful tool for mapping signaling networks in plants, and reveals broad roles of this GSK3-like kinase in cellular signaling and regulation.

INSTRUMENT(S): Q Exactive

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

TISSUE(S): Whole Plant

SUBMITTER: Chuan-Chih Hsu

LAB HEAD: Zhi-Yong Wang

PROVIDER: PXD017085 | Pride | 2023-05-10

REPOSITORIES: Pride

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Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics.

Kim Tae-Wuk TW Park Chan Ho CH Hsu Chuan-Chih CC Kim Yeong-Woo YW Ko Yeong-Woo YW Zhang Zhenzhen Z Zhu Jia-Ying JY Hsiao Yu-Chun YC Branon Tess T Kaasik Krista K Saldivar Evan E Li Kevin K Pasha Asher A Provart Nicholas J NJ Burlingame Alma L AL Xu Shou-Ling SL Ting Alice Y AY Wang Zhi-Yong ZY

The Plant cell 20230301 3

Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-IN ...[more]

PMID: 36660928

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Project description:Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein Tissue inhibitors of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.

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.

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 second question, we used nuclei of developing stomatal guard cells, which express the transcription factor FAMA, as our 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 nuclear TurboID under the FAMA or UBIQUITIN10 (UBQ10) promoter were treated with biotin to label nuclear proteomes. Col-0 wild type was used as controls for unspecific binding of proteins to the beads. Biotinylated proteins were isolated by affinity purification with streptavidin-coupled beads and identified by LC-MS/MS. By targeting TurboID to the nucleus we were able to purify nuclear proteins with relatively high specificity. Enrichment of FAMA-stage specific proteins, when nuclear TurboID was driven by the FAMA compared to the UBQ10 promoter, supports general applicability of TurboID for identification of subcellular proteomes.

Project description:S23 experiment: We sought to identify the microRNAs (miRNAs) enriched in the neural crest cell (NCC) population from E11.5 mouse embryos. To accomplish this, we utilized a transgenic mouse line harboring Cre-recombinase under the control of the Wnt1 NCC-specific promoter and also carrying the R26R-YFP allele. We sorted YFP+ (NCCs) and YFP- (non-NCCs) from E11.5 Wnt1Cre-R26R mouse embryos via FACS and compared the relative enrichment of miRNAs in the YFP+ population by miRNA microarray. 220 experiment: We sought to identify the microRNAs (miRNAs) enriched in the neural crest cell (NCC) population from E10.5 mouse embryos. To accomplish this, we utilized a transgenic mouse line harboring Cre-recombinase under the control of the Wnt1 NCC-specific promoter and also carrying the R26R-YFP allele. We sorted YFP+ (NCCs) and YFP- (non-NCCs) from E10.5 Wnt1Cre-R26R mouse embryos via FACS and compared the relative enrichment of miRNAs in the YFP+ population by miRNA microarray. 221 experiment: Our research has shown that heterozygous deletion of the miRNA processing enzyme Dicer leads to developmental delay of the thymus in mouse embryos. We sought to identify the microRNAs (miRNAs) affected by the loss of a single copy of Dicer in the neural crest cell (NCC) population from E10.5 mouse embryos. To accomplish this, we utilized a transgenic mouse line harboring a floxed allele of Dicer, Cre-recombinase under the control of the Wnt1 NCC-specific promoter, and also carrying the R26R-YFP allele. We sorted YFP+ (NCCs) cells from E10.5 Dicerfl/+,Wnt1Cre,R26R and Dicer+/+,Wnt1Cre,R26R mouse embryos via FACS and compared the relative expression of miRNAs in the Dicer-heterozygotes compared to Dicer-wildtypes by miRNA microarray. S23 experiment: RNA from YFP+ (NCCs) and YFP- (non-NCCs) cells sorted by FACS from E11.5 Wnt1Cre-R26R mouse embryos was isolated and hybridized to Exiqon miRNA microarrays v10. Cells from five E11.5 embryos were sorted into YFP+ and YFP- populations and pooled. 220 experiment: RNA from YFP+ (NCCs) and YFP- (non-NCCs) cells sorted by FACS from E10.5 Wnt1Cre-R26R mouse embryos was isolated and hybridized to Exiqon miRNA microarrays v10. Cells from ten E10.5 embryos were sorted into YFP+ and YFP- populations and pooled. 221 experiment: RNA from YFP+ (NCCs) cells sorted by FACS from E10.5 Dicerfl/+,Wnt1Cre,R26R and Dicer+/+,Wnt1Cre,R26R mouse embryos was isolated and hybridized to Exiqon miRNA microarrays v10. Cells from ten embryos were sorted into YFP+ populations and pooled for each genotype.

Project description:Background: The carpel margin meristem is a vital multi-potent structure located in the medial domain of the Arabidopsis thaliana gynoecium, the female floral reproductive organ. The carpel margin meristem generates ovules that upon fertilization become seeds. The molecular mechanisms that specify this meristematic region and regulate its organogenic potential are poorly understood. Here, we present an analysis of the transcriptional profile of the medial domain of the Arabidopsis gynoecium highlighting the developmental stages that immediately proceed ovule initiation, the earliest stages of seed development. Results: Using a floral synchronization system and a SHATTERPROOF2 domain-specific reporter construct, paired with fluorescence-activated cell sorting, we assayed the transcriptome of the gynoecial medial domain with temporal and spatial precision. Our analysis reveals a set of genes that are differentially-expressed within the SHATTERPROOF2 expression domain that marks portions of the developing medial domain. Many members of this gene set have been shown previously to function during the development of medial domain-derived structures, including the ovules, thus validating our approach. Other uncharacterized members of this gene set, including a set of differentially-expressed cis-natural antisense transcripts, are potential novel regulators of medial domain development and candidates for future functional studies. Several members of the REM family of transcriptional regulators were enriched in the SHP2-expressing cell population including a previously unrecognized REM family member (At5g60142). Analysis of the abundance of specific transcriptional isoforms identified genes that may exhibit âisoform switchingâ behavior during gynoecial development. Conclusions: This data set provides genome-wide transcriptional insight into the development of the gynoecial medial domain that contains the carpel margin meristem, an important reproductive structure that gives rise to the ovules in Arabidopsis thaliana. Four samples (YFP-positive, YFP-negative, all-sorted, and non-sorted). Four biological replicates of each sample (except YFP-positive, collected in triplicated). Strand-specific libraries were sequenced (single-end) in two lanes of a HiSeq 2500 Illumina flow cell (each lane was later analyzed as a technical replicate).

			Action	DRS
	Experimental_Design.xlsx	Xlsx
	GFP-IP-B14Y15-forward.txt	Txt
	GFP-IP-Y14B15-reverse.txt	Txt
	Q20190712_04.mgf	Mgf
	Q20190712_04.mzML	Mzml