Proximity labeling and identification of endogenous client proteins recruited to Y15‐based artificial granules tethering a bait protein

Protein clustering is a ubiquitous event in diverse cellular processes. Self‐association of proteins triggers recruitment of downstream proteins to regulate cellular signaling. To investigate the interactions in detail, chemical biology tools to identify proteins recruited to defined assemblies are required. Here, we exploit an identification of proteins recruited in artificial granules (IPRAG) platform that combines intracellular Y15‐based supramolecule construction with a proximity labeling method. We validated the IPRAG tool using Nck1 as a target bait protein. We constructed Nck1‐tethering granules, labeled the recruited proteins with biotin, and analyzed them by LC‐MS/MS. As a result, we successfully identified proteins that directly or indirectly interact with Nck1.


| INTRODUCTION
Protein clustering underlies various intracellular events.Selfassociation of proteins triggers the formation of complexes with downstream proteins to activate signaling pathways. 1For example, the oligomerization of LAT protein increases Grb2 protein dwelling time, activating intracellular signal transduction. 2,3Liquid-liquid phase separations (LLPSs) are also thought to be initiated by self-association of scaffolding proteins. 4,5Subsets of client proteins are recruited to these transient assemblies and activate membraneless compartments in response to specific events such as cell stress, 6 the cell cycle, 7 or external conditions. 8tificial assembling platforms are promising approaches for studying intracellular protein-protein interactions and the cellular functions of their clusters.Conventionally, antibody-mediated clustering of receptors is widely used to study the effects on Abbreviations: AA, acrylamide; AG, azamigreen; Cry2, cryptochrome 2; DMEM, Dulbecco's modified Eagle medium; DMSO, dimethyl sulfoxide; FUS, fused in sarcoma; G3BP1, GTPaseactivating protein SH3 domain-binding protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Grb2, growth factor receptor-bound protein 2; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; HRP, horseradish peroxidase; IPRAG, identification of proteins recruited in artificial granules; LAT, linker for activation of T cells; LLPS, liquid-liquid phase separation; N-WASP, neural Wiskott-Aldrich syndrome protein; PRM, proline-rich motif; RIPA, radioimmunoprecipitation assay; SAP, self-assembling peptide; SH2, Src homology 2; SH3, Src homology 3; Tks4, SH3 and PX domain-containing protein 2B.3][14][15] Recently, our group has developed a Y15 peptide tag (YEYKYEYKYEYKYEY) for the artificial clustering of proteins in cells. 16,17Y15 is a 15-residue self-assembling peptide (SAP) consisting of hydrophobic Tyr and hydrophilic Glu and Lys repeats and Y15-tagged proteins form clusters in mammalian cells.By tagging the Y15 peptide with the tetrameric fluorescent protein Azamigreen (AG), a micrometer-sized granule is constructed through multivalent interactions.We can decorate the Y15-AG granule by coexpression with Y15-tagged proteins of interest.Furthermore, based on SAP-tag methods, we have developed a client protein enrichment assay that can analyze protein-protein interactions by tethering a bait protein on granules and monitoring the client protein enrichment. 18 uncover the details of protein-protein interactions accompanied by protein clustering, it is important to identify what proteins are recruited into the artificially defined clusters.Proximity labeling techniques are powerful tools to analyze protein interaction networks. 19,20promiscuous labeling enzyme, such as TurboID, is genetically fused to a target protein to label the neighboring proteins. 21The labeled proteins are harvested and identified by LC-MS/MS analysis.[24] In this study, we have developed an identification of proteins recruited in artificial granules (IPRAG) platform by a combination of the Y15 peptide tag technique with proximity labeling.Y15 tag tools have enabled us to design and construct defined granules decorated with a bait protein and TurboID, a promiscuous biotin ligase, in living cells.Endogenous proteins recruited into the granules were biotinylated by TurboID and analyzed by LC-MS/MS.
We have demonstrated the validity of this IPRAG approach by constructing Nck1 clusters that are involved in actin polymerization signaling.The results reveal that this technique can identify not only proteins directly interacting with Nck1 but also proteins involved in actin polymerization signaling.The IPRAG platform offers fundamental tools to reveal the relationships between a bait cluster and protein recruitment.

| Confocal microscopy observation
The transfected cells on a 96-well glass plate were observed two days after transfection.The media was substituted with D-MEM (HEPES, no Phenol Red, Wako) before observation.Imaging was performed using confocal laser fluorescence microscopy (LSM780, Zeiss) with ZEN2.3 (black) software.mTagBFP2, AG, mCherry, and miRFP670 fluorescence signals were detected using a 405-nm laser diode, a 488-nm argon laser, a 561-nm diode-pumped solid-state laser, and a 633-nm He-Ne laser for excitation.The partition coefficient of hit proteins was calculated by using Python 3.8.5 as in the previously reported methods. 18The number of cells forming granules was counted manually by observing 50 transfected cells in each replicate.The experiments were triplicated on different dates.

| Biotinylation of proteins recruited into the bait-tethered granule
Two days after transfection, 100 mM biotin stock in DMSO was added to DMEM to make a final concentration of 50 μM, and the transfected cells were cultured in the biotin-containing medium at 37 C for 1 h.After being washed five times with ice-cold PBS, cells were lysed with RIPA buffer (Nacalai) containing 0.2% SDS and 1% protease inhibitor cocktail (Nacalai) by gentle pipetting.The cell lysate was centrifuged (13,700Âg, min, 5 min, 4 C), and the supernatants were collected.The protein concentrations were quantified by bicinchoninic acid (BCA) assay (Takara).Tris-HCl (pH 9.0).

| Western blotting
To competitively elute the biotinylated proteins from the streptavidin beads, 3 Â Laemmli buffer (150 mM Tris-HCl, 30% glycerol, 6% SDS, 0.01% bromophenol blue, 100 mM dithiothreitol, pH 6.8) with 2 mM biotin was added to the beads, followed by boiling at 95 C for 10 min, and then diluted with ultrapure water.Otherwise, samples were diluted by adding 2 Â Laemmli buffer and boiling at 95 C for 10 min.
After washing three times with TBS-T, the membranes were soaked in 1% skimmed milk in TBS-T with a goat anti-mouse IgG-HRP conjugate (Abcam, ab97265, 1:2,000 dilution) or goat anti-rabbit IgG-HRP conjugate (Fisher Sci, Product#32460, 1:2,000 dilution), incubated for 1 h at room temperature, and washed again three times with TBS-T.Chemiluminescence was induced with Amersham ECL prime reagents (Cytiva) and detected using LuminoGraph I (ATTO).

| Immunostaining
The cells were seeded on 96-well glass plates, where each well was coated with 50 μg/ml fibronectin (Wako).

| LC-MS/MS analysis
After the pull-down of biotinylated proteins with streptavidin beads, the beads were resuspended in 100 μl of urea buffer.Then, 0. were selected to calculate the fold change.The number of peptides was filtered by more than twice in both replicates, and the average of the abundance ratio was calculated.For proteins with an abundance ratio greater than 100, the average was calculated by assuming its value to be 100.

| Strategy for identifying proteins recruited into artificial bait-tethered granules
For identifying client proteins recruited to target granules, we exploited an IPRAG (identification of proteins recruited in artificial granules) technique in combination with our SAP-based granule formation with proximity labeling.In the IPRAG assay, we fabricated artificial three-component granules (Y15-AG-HA, a scaffolding protein; Y15-mTagBFP2-bait, a bait protein; and Y15-mCherry-TurboID-V5, a promiscuous biotin ligase) in cells (Figure 1).Y15-AG-HA selfassembles into granules through the cooperative interactions of Y15 peptide self-assembly and AG tetramer formation. 17 For proof-of-concept of IPRAG, we focused on Nck1-N-WASP-Arp2/3 complex signaling, essential for lamellipodia, and invadopodia formation, which is triggered by clustering of Nck1 (SH2/SH3-containing adaptor protein). 2,25,26Nck1 clustering induced by interaction with phosphorylated residues on membrane receptors via the SH2 domain recruits N-WASPs (neuronal Wiskott-Aldrich syndrome proteins). 27The multivalent interaction between the SH3 domains of Nck1 and the PRMs (proline-rich motifs) of N-WASP leads to phase separation where the Arp2/3 complex is activated, and actin polymerization is promoted. 28Previously, our group has demonstrated the Y15-mediated clustering of SH3 domains of Nck1(1-258) and activate actin polymerization signaling. 17Furthermore, the interaction partners of Nck1 have been well studied, 29 making this suitable for demonstrating the validity of our IPRAG strategy.Hence, we chose the Nck1 SH3 domain (residues 1-258) as the model bait and sought to identify the proteins selectively recruited to the artificial granules.peptides that induce ribosomal skipping during protein translation and enable multiple protein expressions from a single transcript. 30Genes coding Y15-AG-HA, Y15-mTagBFP2-Nck1(1-258), and Y15-mCherry-TurboID-V5 were linked with a self-cleaving 2A peptide sequence (Figure 2A).2][33] The Y15-AG-HA gene was introduced at the first position to secure a high translation level because ribosome drop-off causes a decrease in translation of downstream proteins. 33For a negative control, we also prepared a tricistronic construct coding a non-tagged mTagBFP2-Nck1(1-258) instead of Y15-mTagBFP2-Nck1(1-258) (Figure 2B).
Confocal images of the transfected cells showed the coassembly of the three Y15-tagged proteins (Figure 2C).We noticed that low expression of Y15-AG-HA suppressed the granular formation.In fact, 41.9% ± 2.6% of transfected cells with high expression showed fluorescent puncta (Figure S1).SDS-PAGE and western blotting exhibited bands of each protein at the appropriate molecular weight, indicating that the three proteins were expressed separately (Figure S2).In contrast, the non-tagged Nck1 dispersed throughout the cytosol as expected (Figure 2C).Without Y15-AG-HA, Y15-tagged TurboID and Y15-tagged Nck1 were uniformly Artificial granules decorated with bait (protein of interest) and TurboID are designed and constructed in living cells.The endogenous proteins related to bait clustering are enriched in the granules, which are labeled by TurboID.The biotinylated proteins are purified using streptavidin-coated beads, digested, and identified by LC-MS/MS.distributed in the cytoplasm, suggesting the importance of the scaffolding protein (Figure S3).

| Proximity labeling of enriched proteins in the Nck1(1-258)-tethered granules
We performed proximity labeling by incubating the transfected NIH/3T3 cells with 50 μM biotin and lysed them in RIPA buffer.
We noticed that some biotinylated proteins were detected in insoluble fractions at low (0.1%) SDS concentrations, indicating the granules were not fully dissolved (Figure S4).Therefore, RIPA buffer containing 0.2% SDS was used for the following experiments to obtain the whole biotinylated proteins as soluble forms.Western blotting showed that the band intensity of biotinylated proteins increased with the labeling time and reached a plateau at 120 min (Figure 3A).Considering the time profile, we set the reaction time as 1 h to avoid undesired reactions a decrease in the spatial resolution of the labeling. 21mpared with negative control cells expressing non-tagged Nck1, cells expressing Y15-tagged Nck1(1-258) showed two novel bands with strong intensities (Figure 3B, arrow heads).The pull-down experiment using streptavidin beads also revealed that these bands were markedly different in intensity, suggesting that proteins recruited to the granules were selectively labeled.
Fluorescence imaging of Alexa647-streptavidin-stained cells showed that biotinylated proteins clearly colocalized with the Y15-AG scaffold (Pearson's r = 0.86 ± 0.07), indicating that Y15-tagged TurboID selectively labeled the proteins within Y15-based granules and the granules were maintained even after biotinylation (Figure 3D).digestion for LC-MS/MS (Figure 4A).As a result, 347 proteins were identified in total.These were filtered to those with more than two peptides detected in both biological replicates, yielding 179 proteins (Figure 4B).We identified eight potential hit proteins excluding Nck1 as those with an average abundance relative to the control of greater than 5.0 (Table 1).A list of all the hit proteins is shown in Data S1.A variety of proteins with different molecular weights and isoelectric points (pI) were identified.
Among these eight hit proteins, the top two proteins (N-WASP and Cortactin; abundance ratios over 100) have been reported to interact with Nck1. 27,34,35For validation, fluorescent protein miRFP670 was fused to N-WASP and Cortactin to observe enrichment in the Nck1 cluster.Confocal images showed that both miRFP670-N-WASP and miRFP670-Cortactin were enriched in the Nck1(1-258) cluster, although the enrichment level of Cortactin was relatively low (Figures 4C and S5).Western blotting also revealed that endogenous N-WASP was biotinylated by TurboID and pulled down by streptavidin beads (Figure 4D).
Although Cortactin is reported to bind to an SH2 domain of Nck1, 36 the Nck1(1-258) tested in this study is truncated in the SH2 domain.As an SH3 domain of Cortactin is known to bind to PRMs of N-WASP, this suggests that Cortactin is indirectly enriched in the Nck1(1-258) granules through binding to N-WASP. 34,37Similarly, a Tks4 protein hit in this study (abundance ratio, 15.3) was observed in the co-assembly with Cortactin upon EGF stimulation to form membrane protrusions. 38A direct interaction of Tks4 with N-WASP has been validated by pull-down assay, 39 indicating that our IPRAG technique identifies not only direct interacting proteins but also a variety of proteins associated with the signaling pathway (shown by red crosses in Figure 4B).For proteins with an abundance ratio greater than 100, the average value was calculated by assuming its value to be 100.
N-WASP, which is known to directly interact with Nck1, but also proteins involved in actin polymerization signaling such as Cortactin and Tks4 were identified.In addition, we also identified five proteins with undefined roles in actin polymerization signaling.Further studies are required to understand the relationship between the Nck1 cluster and these proteins.
In the conventional approach, TurboID has been directly fused to a marker protein to identify proteins within certain subcellular compartments. 19However, in order to elucidate mechanisms of protein cluster formation and their following events, it is critical to artificially form well-defined protein clusters in cells and investigate what proteins recruit to these clusters.IPRAG (a bottom-up method in which clusters are artificially formed and evaluated) is a complementary technique to the conventional top-down method.
As a similar approach to IPRAG, a fusion of Cry2 and TurboID to target proteins was reported to identify proteins enriched in artificially induced membraneless compartments. 40,41Light-induced oligomerization of Cry2 triggers phase separation, and TurboID biotinylates the proteins recruited into the condensates, allowing the analysis of the protein composition by LC-MS/MS.This method has high temporal resolution, because light irradiation can induce in situ droplet formation.However, the disadvantage of Cry2 is the complicated operation, which requires precise control of the light intensity and exposure time at appropriate intervals to maintain droplets while paying attention to the generation of reactive oxygen species. 41,42In contrast, the Y15 tag can rationally incorporate bait proteins into the Y15-AG scaffold.Moreover, the small tag size and methodological simplicity of IPRAG are advantages for clustering multiple proteins.In addition, a previously developed guide tag system 18 enables a more detailed analysis of cluster formation in cells.The SAP tag-based toolkits allow in situ protein complex reconstruction with a substantial proteome analysis, providing a platform for elucidating intracellular protein clustering events through a bottom-up approach.
After adding lysate supernatants containing 100 μg proteins to 12 μl of pre-washed Dynabeads MyOne Streptavidin C1 (VERITAS) suspension, the biotinylated proteins were pulled down by rotating the tubes at 4 C overnight.Subsequently, the beads were washed with 400 μl of RIPA buffer, three washes with urea buffer (1 Â TBS [NIPPON GENE], 1 M urea, pH 7.4), three washes with 400 μl of salt buffer (1 Â TBS, 1 M NaCl, pH 7.4), and three washes with 50mM 5 μg of Trypsin/Lys-C mix (Promega, Cat.#V5072) was added and incubated by rotating the tubes at 37 C overnight.The cleaved peptide fragment solution was collected and desalted using GL-Tip™ SDB (GL Science, 7820-11200) following the manufacturer's protocol.The resulting solution was dried by vacuum centrifugation, resuspended in 2% CH 3 CN/0.1% TFA, and subjected to LC-MS/MS analysis.LC-MS/MS analysis was performed by a nanoLC-ESI-MS system, composed of a quadrupole-orbitrap hybrid mass spectrometer (Q-Exactive, Thermo Fisher Scientific) equipped with a nanospray ion source and a nano HPLC system (Easy-nLC 1000, Thermo Fisher Scientific).Each sample was loaded onto a trap column packed with 3 μm C18-silica particles (2 cm Â 75 μm capillary column, Thermo Fisher Scientific) linked with a separation column packed with 3 μm C18-silica particles (12.5 cm Â 75 μm capillary column, Nikkyo Technos).The samples were separated by applying a 10-40% linear acetonitrile gradient over 70 min in the presence of 0.1% formic acid with the flow rate at 300 nl/min.Xcalibur 4.0 (Thermo Fisher Scientific) was operated in datadependent acquisition mode to obtain a full MS scan (resolution: 70,000) and MS2 scan (resolution: 17,500).The AGC target was 3.0 Â 10 6 for a full MS scan and 5.0 Â 10 5 for MS2 scan.The maximum ion time used for a full MS scan and MS2 scan was 60 ms.The full MS scan range was 310-1,500 m/z.For the MS2 scan, the top 10 signals were selected per one full MS scan, and the dynamic exclusion was 15 s.The measurement was performed three times for each sample, and two biological replicates were measured in each condition.Protein identification and quantification were performed by Proteome Discoverer 2.4 software with the Sequest HT search engine (Thermo Fisher Scientific).The Mus musculus protein list was obtained from the UniProt database (UP000000589, downloaded on August 23, 2021).Label-free quantification was performed with the following settings: normalization mode, total peptide amount; protein abundance calculation, summed abundance; protein ratio calculation, protein abundance based; maximum allowed fold change, 100; imputation mode, none; and hypothesis test, ANOVA (individual proteins).Only peptides/proteins with a <1% false discovery rate (Percolator algorithm) and Master annotation of "IsMasterProtein" Bait protein is incorporated into the Y15-based scaffold by Y15 tagging, leading to the recruitment of a subset of endogenous proteins involved with bait clustering.The Y15-tagged TurboID allows selective biotinylation of the proteins recruited into granules, which are identified by conventional proteomic analysis.

F I G U R E 3
Biotin labeling of proteins recruited to Nck1(1-258)-tethered granules by TurboID.(A) Western blotting for time profile of biotin labeling by Y15-tagged TurboID using anti-GAPDH antibody for loading control and an anti-V5 tag antibody to visualize Y15-mCherry-TurboID-V5. (B) Comparison of biotinylated proteins in cell lysates expressing Y15-tagged and non-tagged Nck1(1-258).The biotinylated proteins in whole cell lysate (left panel) and in the pull-down sample (right panel) are blotted by HRP-streptavidin.Arrow heads indicate bands that have strong intensity compared with negative control.(C) Proximity labeling of Y15-tagged Nck1(1-258) in granules.(D) Colocalization of biotinylated proteins with granules in NIH/3T3 cells.The scale bar means 10 μm.In this study, we devised the IPRAG technique with the combination of Y15-based artificial granule formation with proximity labeling to identify proteins recruited to target granules.A Y15-tagged target protein (bait) and Y15-tagged TurboID were incorporated into the Y15-AG scaffold.Recruited endogenous proteins were biotinylated by Y15-tagged TurboID, collected using streptavidin beads, and identified by LC-MS/MS analysis.We demonstrated this strategy using the Nck1-N-WASP-Arp2/3 complex as a model.As a result, not only