{"doi":"10.1111/nph.20014","title":"<scp>TurboID</scp>‐based proteomic profiling reveals proxitome of <scp>ASK1</scp> and <scp>CUL1</scp> of the <scp>SCF</scp> ubiquitin ligase in plants","abstract":"The SKP1-Cullin-F-box (SCF) complex is one of the best-studied E3 ubiquitin ligases in plants because of its critical roles in various signaling pathways (Lechner et al., 2006; Sadanandom et al., 2012; Stefanowicz et al., 2015; Abd-Hamid et al., 2020). The core components of SCF include Cullin 1 (CUL1) scaffold protein that interacts with SKP1 adaptor protein at the N-terminal, and E2-interacting RING-finger protein RBX1 at the C-terminal (Fig. 1a). The SKP1 interacts with interchangeable F-box receptor units that specifically recognize target substrates for ubiquitination and degradation by the 26S proteasome (Zheng & Shabek, 2017). Among the 21 SKP1-like (ASK) proteins in Arabidopsis, 19 exhibit significant structural similarity and are thought to be functionally redundant. Historically, studies have focused on ASK1 due to its prominent function as an adaptor in the SCF module, with higher steady-state levels of expression throughout the plant, particularly in proliferating tissues, compared with other ASK proteins (Porat et al., 1998; Yang et al., 1999; Zhao et al., 1999; Gagne et al., 2002; Risseeuw et al., 2003). The Arabidopsis genome is predicted to encode hundreds of F-box proteins that target thousands of substrate proteins, but the majority remain uncharacterized and without known substrates (Abd-Hamid et al., 2020). Interestingly, mammalian SKP1 has recently been shown to regulate the switch between autophagy and unconventional secretion (Li et al., 2023), suggesting that its biological function extends beyond acting as an adapter protein within the SCF complex. Thus far, identifying E3 substrates has been challenging because substrates often interact weakly and transiently with the E3, and they are rapidly degraded and hence difficult to capture (Pierce et al., 2009; Iconomou & Saunders, 2016). Although traditional methods such as affinity purification coupled to mass spectrometry (AP-MS), yeast-two hybrid (Y2H), and protein microarray screening have captured E3 substrates with some success, each has its own drawbacks (Harper & Tan, 2012; Iconomou & Saunders, 2016). AP-MS fails to capture weak and transient interactors, whereas Y2H is labor intensive, prone to false positives, and is a heterologous system. The recently developed proximity labeling (PL) approach to capture protein–protein interactions (PPI) in vivo overcomes many of the drawbacks of traditional approaches. For PL, a protein of interest (POI) is fused to a promiscuous biotin ligase. This ligase catalyzes biotin to a short-lived biotinoyl-5′-AMP, which can diffuse away from the ligase and react with amine groups on lysine residues of nearby proteins, typically within a radius of 10 nm (Kim & Roux, 2016; Qin et al., 2021; Yang et al., 2021). The biotinylated proximal interactomes (i.e. proxitomes) of the POI can be enriched using streptavidin-conjugated beads under stringent conditions followed by proteome identification through mass spectrometry (MS; Kim & Roux, 2016; Qin et al., 2021; Yang et al., 2021). In this study, we utilized the TurboID biotin ligase, which is highly efficient at transferring biotin to proximal proteins in many organisms, including plants (Branon et al., 2018; Mair et al., 2019; Zhang et al., 2019), to identify the SCF interactome in Arabidopsis. We uncovered the CUL1 and ASK1 proxitomes, providing insights into the functions of interactors in both conventional SCF-mediated signaling pathways and potentially noncanonical SCF-independent processes. Further investigation and analysis of the interactomes associated with ASK1 and CUL1 revealed novel partners involved in diverse biological processes within plants. The TurboID-based PL strategy detailed in this study can be expanded to identify targets belonging to other E3 families in plants. This expansion would enhance the repertoire of components within the ubiquitin-proteasome system and shed light on their pivotal roles in regulating a multitude of cellular processes. Arabidops","journal":"New Phytologist","year":2024,"id":434262,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":13,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.955,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1240420,"name":"Natalie Hamada","orcid":"0000-0003-0388-5177","position":1,"is_corresponding":false},{"id":173936,"name":"Christian Montes","orcid":"0000-0003-1249-2308","position":2,"is_corresponding":false},{"id":471651,"name":"Yuanyuan Li","orcid":"0000-0001-8615-4374","position":3,"is_corresponding":false},{"id":770223,"name":"Nathan Meier","orcid":"0000-0003-2312-0059","position":4,"is_corresponding":false},{"id":173938,"name":"Justin W Walley","orcid":"0000-0001-7553-2237","position":5,"is_corresponding":false},{"id":94452,"name":"Savithramma P. Dinesh‐Kumar","orcid":"0000-0001-5738-316X","position":6,"is_corresponding":false},{"id":853229,"name":"Nitzan Shabek","orcid":"0000-0002-2190-5955","position":7,"is_corresponding":false},{"id":956820,"name":"Fuai Sun","orcid":"0000-0003-4076-661X","position":0,"is_corresponding":true}],"reference_count":51,"raw_metadata":null,"created_at":"2026-07-19T01:59:53.957667Z","pmid":"39081016","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}