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

Abstract

1Transient protein-protein interactions (PPIs), such as those between posttranslational 2 modifying enzymes and their substrates, play key roles in cellular regulation, but are difficult 3 to identify. Here we demonstrate the application of enzyme-catalyzed proximity labeling (PL), 4 using the engineered promiscuous biotin ligase TurboID, as a sensitive method for 5 characterizing PPIs in signaling networks. We show that TurboID fused with the GSK3-like 6 kinase BIN2 or a PP2A phosphatase biotinylates their known substrate, the BZR1 transcription 7 factor, with high specificity and efficiency. We optimized the protocol of biotin labeling and 8 affinity purification in transgenic Arabidopsis expressing a BIN2-TurboID fusion protein. 9Subsequent quantitative mass spectrometry (MS) analysis identified about three hundred 10 proteins biotinylated by BIN2-TurboID more efficiently than the YFP-TurboID control. These 11 include a significant subset of previously proven BIN2 interactors and a large number of new 12 BIN2-proximal proteins that uncover a broad BIN2 signaling network. Our study illustrates 13 that PL-MS using TurboID is a powerful tool for mapping signaling networks, and reveals 14 broad roles of BIN2 kinase in cellular signaling and regulation in plants. 16Protein-protein interactions (PPIs) organize proteins into cellular structures, metabolic 18

Establishment of Proximity-dependent Biotinylation Approaches in Different Plant Model Systems

“…Our results are complementary to the work deposited in BioRxiv reporting the use of TurboID to identify transient signalling components (Kim et al, 2019) and novel regulators of plant immunity (Zhang et al, 2019), as well as for the efficient capturing of cell- and subcellular compartment-specific interactomes (Mair et al, 2019). Taken together, these four studies provide a new arena for the identification of novel protein-protein interactions in plants.…”

Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions

“…A homozygous 35S::AtACINUS-GFP / acinus-2 plant was selected for similar protein expression level to the endogenous AtACINUS protein of wild-type plants using a native α-AtACINUS antibody, and crossed with acinus-2 pinin-1 to obtain 35S::AtACINUS-GFP / acinus-2 pinin-1 transgenic lines. Similarly, 35S::AtACINUS-YFP-TurboID plasmid was generated by LR reaction of gateway-compatible 35S::YFP-TbID 65 with pENTR-AtACINUS and transformed to acinus-2 pinin-1 to obtain transgenic lines.…”

“…For affinity purification of AtACINUS using in vivo biotinylation, the acinus pinin mutant was transformed with a T-DNA construct that expresses AtACINUS as a fusion with TurboID from the 35S promoter 65 . The AtACINUS-YFP-TurboID / acinus-2 pinin-1 seedlings were treated with 0 or 50 μmmol/L biotin for 3 hours.…”

“…The AtACINUS-YFP-TurboID / acinus-2 pinin-1 seedlings were treated with 0 or 50 μmmol/L biotin for 3 hours. The AtACINUS-YFP-Turbo protein was affinity purified using streptavidin beads as previously described 65 using a modified extraction buffer containing 20 μmol/L PUGNAC and 1 x PhosphoStop. After on-bead tryptic digestion, the samples were analyzed as described above in the label-free IP-MS section on a Q-Exactive HF instrument.…”

Proximity‐dependent labeling methods for proteomic profiling in living cells: An update

“…Many groups have applied proximity labeling tools in developmental systems, such as Arabidopsis (Khan, Youn, Gingras, Subramaniam, & Desveaux, 2018;Kim et al, 2019;Mair et al, 2019), C. elegans (Branon et al, 2018;Reinke, Mak, Troemel, & Bennett, 2017), Drosophila (Branon et al, 2018;Chen, Hu, et al, 2015;Li et al, 2020;Mannix et al, 2019;Shinoda, Hanawa, Chihara, Koto, & Miura, 2019), and mouse (Brudvig et al, 2018;Dingar et al, 2015;Uezu et al, 2016). Importantly, some have used proximity-labeling tools to discover new components of developmental processes.…”