<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. Arabidopsis thaliana Col-0 and Nicotiana benthamiana plants were grown at 22°C in a growth chamber with a 16 h : 8 h light : dark photoperiod. The Agrobacterium tumefaciens (Agrobacterium) strain GV3101, containing pUBQ:TurboID-3xMyc-CUL1NTD/gASK1 and pUBQ:Citrine-TurboID-3xMyc, was used to generate stable transgenic Arabidopsis lines by floral-dip method. For bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), degradation, and ubiquitination assays, the GV3101 cultures carrying each construct were adjusted to an OD600 of 1. The appropriate cultures were combined in a 1 : 1 ratio and infiltrated into N. benthamiana with the infiltration buffer (10 mM MgCl2, 10 mM MES pH 6, and 250 μM acetosyringone). MG132 (50 μM) was infiltrated 12 h before tissue collection. For the TurboID experiment, the N-terminal domain (NTD) of AtCUL1 (1–380 aa), genomic DNA of AtASK1 (gASK1), and citrine were amplified via polymerase chain reaction. Both polymerase chain reaction fragments and vector were digested with XmaI and MluI enzymes to create pUBQ:TurboID-3xMyc-CUL1NTD/gASK1 and pUBQ:citrine-TurboID-3xMyc constructs under the control of the Arabidopsis ubiquitin promoter (pUBQ) and nopaline synthase (NOS) terminator (Supporting Information Table S1a). The vector contains a kanamycin resistance gene for selection in E. coli and a BASTA resistance gene for selection in Arabidopsis. For BiFC, Co-IP, degradation, and ubiquitination assays, polymerase chain reaction fragments were amplified for the generation of constructs p35S:YC3xHA-CUL1NTD/gASK1 and p35S:targets-YN3xMyc under the control of cauliflower mosaic virus 35S promoter (p35S) and NOS terminator using the Gateway cloning method (Invitrogen). Additionally, for the ubiquitination assay, the initial 76 amino acids of the coding sequence of AtUBQ10 (AT4G05320) were amplified to create pUBQ:3xHA-Ubiquitin by infusion method (In-Fusion Snap Assembly cloning kit; Takara, San Jose, CA, USA). All primers used in this study are listed in Table S1(b). For TurboID sample preparation for MS, an adapted version of the protocol from Zhang et al. (2019) was used with three biological replicates. Briefly, the stable transgenic lines were syringe-infiltrated with biotin and incubated under previously determined conditions. Leaves were collected in bulk, flash-frozen, and homogenized into a fine powder using mortar and pestle. For the total protein extraction, 1 g of homogenized tissue was aliquoted per replicate and rotated at 4°C for 30 min with RIPA lysis buffer (50 mM Tris pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% NP40 (v/v), 0.1% SDS (w/v), 0.5% deoxycholate, 1 mM DTT, and protease inhibitor cocktail (Roche)). Tubes were subsequently centrifuged at 16 500 g for 10 min, and the soluble fraction was loaded into a Zeba™ Spin Desalting Columns (Thermo Fisher Scientific, Waltham, MA, USA), 7K MWCO to remove free biotin from the lysate. The concentration of the desalted protein samples was measured by the Bradford method (Kruger, 2009). To enrich biotinylated proteins, 200 μl Dynabeads™ MyOne™ Streptavidin C1 (Thermo Fisher Scientific) were prewashed with RIPA buffer and subsequently loaded with 6 mg desalted protein extract. Extract and beads were rotated at 4°C for 16 h and subsequently washed at room temperature with 1.7 ml buffer I (2% SDS), 1.7 ml buffer II (50 mM HEPES pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% deoxycholic acid, and 1% Triton X-100), and 1.7 ml buffer III (10 mM Tris pH 7.4, 250 mM LiCl, 1 mM EDTA, 0.1% deoxycholic acid, and 1% NP40). The beads were then moved to 4°C, transferred to a new tube, and washed with 1.7 ml 50 mM Tris pH 7.5 and then 6× with 1 ml 50 mM ammonium bicarbonate. After the final wash, 30 μl bead resuspension was collected from each technical replicate for immunoblot analysis. Finally, the ammonium bicarbonate supernatant was removed, and the beads were flash-frozen in liquid nitrogen and stored at −80°C until LC-MS/MS analysis. Enriched samples were eluted from beads by incubation at 95°C for 10 min in 1× S-Trap lysis buffer (5% SDS, 50 mM TEAB, pH 8.5) supplemented with 12.5 mM biotin. Eluted samples were subjected to S-Trap sample processing technology (ProtiFi, Fairport, NY, USA), following the manufacturer's protocol. Samples were reduced in 2 mM TCEP, alkylated in 50 mM iodoacetamide (IAM), and digested into peptides at 37°C in one round of overnight incubation with 1 μg of trypsin and a second incubation of 4 h with 0.1 μg trypsin plus 0.1 μg Lys-C. Peptides were further desalted using SepPack C18 columns (Waters, Milford, MA, USA). Tandem Mass Tag (TMT; Thermo Fisher Scientific) labeling was performed on purified peptides from each sample as reported previously (Song et al., 2020). TMT labeling reaction was stopped using 5% hydroxylamine, and the quenched samples were then pooled. Pooled samples were subjected to high pH fractionation using Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific) following the manufacturer's instructions. The obtained eight fractions were further concatenated (pooled) as follows: Fraction 1 with Fraction 5, Fraction 2 with Fraction 6, Fraction 3 with Fraction 7, and Fraction 4 with Fraction 8. Samples were finally dried on a SpeedVac and resuspended in 0.1% Optima™ grade formic acid (Fisher) in Optima™ grade H2O (Fisher). An injection volume of 20 μl, containing 1.2 μg from each concatenated fraction, was used for LC-MS/MS analysis. Chromatography was performed on a Thermo UltiMate 3000 UHPLC RSLCnano. Peptides were desalted and concentrated on a PepMap100 trap column (300 μM i.d. × 5 mm, 5 μm C18, and 100 Å μ-Precolumn (Thermo Fisher Scientific)) at a flow rate of 10 μl min−1. Sample separation was performed on a 200 cm Micro-Pillar Array Column (μPAC, Pharmafluidics) with a flow rate of c. 300 nl min−1 over a 150 min reverse phase gradient (80% ACN in 0.1% FA from 1% to 15% over 5 min, 15% to 20.8% over 20 min, from 20.8% to 43.8% over 80 min, and from 43.8% to 99.0% in 11 min, and kept at 99.0% for 5 min). Eluted peptides were analyzed using a Q-Exactive Plus high-resolution quadrupole Orbitrap mass spectrometer, which was directly coupled to the UHPLC. Data-dependent acquisition was obtained using the Xcalibur v.4.0 software in positive ion mode with a spray voltage of 2.3 kV and a capillary temperature of 275°C and an RF of 60. MS1 spectra were measured at a resolution of 70 000, an automatic gain control (AGC) of 3e6 with a maximum ion time of 100 ms and a mass range of 400–2000 m/z. Up to 15 MS2 were triggered at a resolution of 35 000. A fixed first mass of 115 m/z, an AGC of 1e5 with a maximum ion time of 50 ms, a normalized collision energy of 33, and an isolation window of 1.3 m/z were used. Charge exclusion was set to unassigned, 1, 5–8, and > 8. MS1 that triggered MS2 scans were dynamically excluded for 25 s. Raw data were analyzed using MaxQuant v.2.1.0.0 (Cox & Mann, 2008). Spectra were searched, using the Andromeda search engine, against Arabidopsis thaliana TAIR10 annotation (Cox et al., 2011; Berardini et al., 2015). The proteome files were complemented with reverse decoy sequences and common contaminants by MaxQuant. Carbamidomethyl cysteine was set as a fixed modification while methionine oxidation and protein N-terminal acetylation were set as variable modifications. The sample type was set to ‘Reporter Ion MS2’ with ‘TMT18plex’ selected for both lysine and N-termini. TMT batch-specific correction factors were configured in the MaxQuant modifications tab (TMT Lot No.: XA338617). Digestion parameters were set to ‘specific’ and ‘Trypsin/P;LysC’. Up to two missed cleavages were allowed. A false discovery rate, calculated in MaxQuant using a target-decoy strategy, < 0.01 at both the peptide spectral match and protein identification level was required (Elias & Gygi, 2007). The match between runs feature of MaxQuant was not utilized. ASK1 and CUL1NTD significant interactors were assessed using the TMT-NEAT R pipeline with the poissonseq R package (Clark et al., 2021; Li et al., 2012). A q-value < 0.1 and log2(fold change) > 0.3 were used as cutoffs for designating enrichment. Significant enrichment of Biological Processes (BPs) and cellular components ASK1 and CUL1NTD interactors was determined using the on et al., et al., 2009). with < were selected as of interactors was on proteins were obtained from of known proteins, plus protein that was of the following protein protein protein ubiquitination The ubiquitin and of and are as ubiquitin and interactors were obtained from and Arabidopsis in The for et al., & 2016). This on and were using containing the POI were and subjected to and then transferred to using a system were with 5% in in for 1 h at room followed by incubation with appropriate at 4°C The the was washed three with 0.1% followed by a incubation with a used in this study include 1 : 1 : 1 : and 1 : were using (Thermo Fisher Scientific) (Thermo Fisher were using a The was using the of CUL1NTD and ASK1 with their targets was as previously et al., 2020). In N. benthamiana with the targets were collected and into a fine powder using liquid The powder was transferred into a tube, and buffer mM pH 7.5, 1 mM EDTA, 150 mM NaCl, 10 mM 1 mM and protease inhibitor cocktail was The was and for 30 min, followed by a for 10 min at beads were with buffer buffer without and were rotated at 4°C for 3 The supernatant was using a and the beads were washed three with a The beads were then to a sample buffer and for 5 min for immunoblot analysis. All and are in Nicotiana benthamiana were using a with an 1 × 1 cm were to the infiltration All within the were under and using and fluorescence were under and The N. benthamiana infiltrated with targets were infiltrated with MG132 (50 12 h before collection. The collected were into a fine powder using liquid sample buffer was directly to the followed by for 5 min for immunoblot analysis. For the ubiquitination assay, Agrobacterium strain GV3101 containing and was into N. 50 μM MG132 was and tissue was collected at The was performed as The target proteins were enriched using beads and for ubiquitination analysis by In the SCF CUL1 with and ASK1 F-box proteins with their target substrates for degradation (Fig. 1a). we used Arabidopsis CUL1 and ASK1 as to capture SCF targets under growth conditions (Fig. 1a). To that we fused and TurboID to the of ASK1 under the control of Arabidopsis ubiquitin promoter (pUBQ) and NOS terminator (Fig. and we the of CUL1 fused to The CUL1NTD was as previously (Zheng et al., to enrich and E3 targets by the and of a control to for we used protein fused to constructs were into Arabidopsis Col-0 and lines were selected and the and for 4 and transgenic lines to Col-0 plants (Fig. The expression of TurboID proteins in lines was by immunoblot analysis (Fig. Among we for biotin infiltration of biotin into biotin and tissue compared with infiltration and in the biotin time and with streptavidin-conjugated revealed that 50 μM biotin infiltration and incubation for at room temperature was for efficient labeling for TurboID (Fig. In Col-0 infiltration of 200 μM biotin followed by incubation at 3 h at room temperature in of biotinylated proteins (Fig. that proteins were in the transgenic lines TurboID To identify ASK1 and CUL1NTD proximal proteins, we performed affinity purification of biotinylated proteins in three (Fig. the and for analysis the enrichment of biotinylated proteins on beads (Fig. The biotinylated proteins to beads were eluted using SDS supplemented with 12.5 mM biotin. Eluted proteins were and The peptides were with TMT and analyzed by liquid mass spectrometry analysis a total of proteins and Biological from plants the a high Among the proteins, and were ASK1 and CUL1 interactors, on the of q-value 0.1 and (Fig. Table between ASK1 and CUL1 interactors common interactors (Fig. Table The and common interactors include F-box proteins and other proteins (Fig. To the of enriched ASK1 and CUL1 we performed enrichment analysis. A of CUL1 interactors were to protein and gene expression (i.e. and Table ASK1 interactors in groups various enriched For both the protein to as was enriched (Fig. Table To further interactors from we a of F-box proteins and proteins to ubiquitination and for in significant enrichment of proteins ASK1 interactors and CUL1 interactors Table In the CUL1NTD interactors, previously were the highly include 3 1 1 and et al., et al., 2002; et al., 2012; et al., 2009; et al., Table The and were highly enriched in the ASK1 data as as A 1 1 5 and 1 which have been previously to 26S degradation but were not specifically shown to be by SCF E3 complex & et al., et al., 2006; et al., & that proteins in are under proteins and 12 previously interactors of ASK1 and CUL1 (Fig. Table we that ASK1 and CUL1NTD TurboID captured a of SCF substrates and of which are To are target substrates of the SCF E3 ligase, we selected a proteins for further in we performed a to the interactions using transient expression in N. benthamiana followed by For we interactors fused to the N-terminal amino acid residues of citrine and CUL1NTD ASK1 fused to the C-terminal amino acid residues of citrine of with citrine but not with control of with citrine but not with control (Fig. Although was as an in PL, we were to citrine fluorescence in the (Fig. that 11 of the 12 proteins in the in we selected a of interactors as novel SCF substrates for the To that fused and were each in N. benthamiana with fused to beads were utilized for the followed by with analysis a of by targets from the (Fig. citrine as a control and not ASK1 (Fig. Additionally, in a experiment, and were by CUL1NTD using beads (Fig. To the are SCF E3 ubiquitin ligase target substrates that ubiquitination and degradation, ubiquitin was with and in N. beads were used to followed by immunoblot using analysis high for each of the substrates (Fig. the of substrates is typically degradation, we the of a proteasome inhibitor & on the degradation of protein levels for substrates were in the of MG132 (Fig. further that the proteins as SCF ubiquitin ligase we the SCF interactome in and new targets of the ubiquitin system by an PL strategy combined with protein and has been previously that and degradation & & 2023), they have not been specifically to SCF ubiquitin ligase complex. is that targets of the ubiquitin system can be by E3 ligase For while is as an SCF target in study, has recently been to ubiquitination by the E3 & is that the of can be by potentially on factors such as expression other such as degradation, a pivotal in has been by the ubiquitin-proteasome as by various for ubiquitination & an of an E3 ligase, and studies are to further the associated F-box and have not been previously shown to and were as and proteins that the of the acid Zhang et al., et al., 2015). ubiquitination of is critical for and of resistance et al., 2019; et al., 2020). This a between SCF ubiquitination and further the ubiquitin system as a in further into the degradation of and would have for as for and has been shown to levels and resistance to et al., 2015; Zhang et al., et al., 2021). a of F-box proteins, the highly efficient of which as an control for its expression and We can that the of F-box proteins be due to in the to capture some F-box proteins that into SCF in tissues, plants are to and is that is by the of lysine residues to which biotin is transferred of the be to selection of ASK1 as a the for ASK1 is by of its prominent as an SCF be that other ASK proteins, such as and in the SCF complex and for F-box selection in tissues, under and at et al., 2002; Risseeuw et al., 2003). is that Arabidopsis ASK1 has mammalian SKP1 has been shown to regulate a switch between autophagy and unconventional protein suggesting that ASK1 have biological functions beyond its as an adaptor protein within the SCF complex (Li et al., the F-box proteins and other proteins we captured under conditions with ASK1 a at a be of noncanonical SCF Further studies transgenic lines be for F-box proteins that into SCF and other targets that with ASK1 in an SCF-independent complex under various biological conditions. In study has revealed novel interactors and substrates of ubiquitin ligase, the pivotal of enzymes in for growth and not the SCF interactome but the for further of the functions of proteins within biological a for the E3 interactome has the to enhance of protein through ubiquitination and degradation in to various and is by the and and are by the of of Biological and and are by from the of and from the of and the is by the of and and the and performed of the in of TurboID assay, and transient expression performed performed LC-MS/MS and analysis. and performed data analysis and and the of the and and the Mass spectrometry data can be from using the and used in this TurboID-based proximity labeling and between samples used for ASK1 and CUL1NTD proximity labeling of between ASK1 and CUL1NTD with Table and proximity labeling sequences used in this Table CUL1 and ASK1 TurboID-based proximity labeling Table ASK1 and CUL1 common Table Table of enriched F-box proteins and Table analysis Table enrichment. Table for is not for the of Information by the be to the The is not for the of by the be to the for the

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