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2B). Overexpression of FBXW1 also increased the ubiquitination of NFKBIA (16) and PDCD4 (17), known. FBXW1 substrates, even in the absence of stimuli (Fig.
A comprehensive method for detecting ubiquitinated substrates using TR-TUBE Yukiko Yoshidaa,1, Yasushi Saekib, Arisa Murakamia,b, Junko Kawawakia, Hikaru Tsuchiyab, Hidehito Yoshiharab, Mayumi Shindoc, and Keiji Tanakab,1 a

Protein Metabolism Project, bLaboratory of Protein Metabolism, and cCenter for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan Edited by Aaron Ciechanover, Technion-Israel Institute of Technology, Bat Galim, Haifa, Israel, and approved March 10, 2015 (received for review November 21, 2014)

The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-e-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE–expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein. ubiquitin-binding protein

of ubiquitinated proteins, has been developed for global proteomic applications aimed at identifying ubiquitinated substrates (8, 9). Although a few quantitative proteomics studies have identified a particular ubiquitin ligase substrate using stable isotope labeling utilizing amino acids in cell culture and the antidiGly antibody (10), these examples required large quantities of samples and advanced techniques. Tandem ubiquitin-binding entity(ies) (TUBE) based on ubiquitinassociated domains have been developed for isolation of polyubiquitinated proteins from cell extracts (11). Notably, TUBE reagents protect polyubiquitin-conjugated proteins in cell lysates from both proteasomal degradation and deubiquitinating enzymes (DUBs) as efficiently as specific inhibitors of these enzymes (11). In this paper, we applied the TUBE technology to in vivo capture of ubiquitinated proteins. To develop a versatile method for identifying substrates of a specific ubiquitin ligase, we designed a mammalian expression vector encoding a FLAGtagged trypsin-resistant (TR) TUBE, which protects ubiquitin chains from trypsin digestion under native conditions. Using two enrichment methods, TR-TUBE and the anti-diGly antibody, we succeeded in identifying the target substrates of the uncharacterized F-box protein FBXO21. Results Protection of Polyubiquitin Chains on Substrates by TR-TUBE. Our

method is based on stabilization of ubiquitinated substrates in vivo by masking of ubiquitin chains with exogenously expressed

| ubiquitin ligase | ubiquitination

Significance

P

osttranslational modification by ubiquitin regulates diverse processes in cells (1, 2). Ubiquitination is catalyzed by three types of enzymes—E1, E2, and E3, with the selectivity for the target protein provided by E3 ubiquitin ligases. Although the human genome encodes more than 600 ubiquitin ligases, many of them remain to be studied (3). The Skp1–Cul1–F-box protein (SCF) complex, one of the best-characterized ubiquitin ligases, is composed of three invariable components (Skp1, Cul1, and Rbx1) and a variable component F-box protein that serves as the substrate recognition module. Among the over 70 F-box proteins found in humans, less than half have been characterized (4). The identification of substrates for a specific ubiquitin ligase has been challenging despite considerable efforts. To date, the physical interaction between an ubiquitin ligase and its substrates has been exploited as the major approach for substrate identification (5–7). In these studies, immunoprecipitation followed by MS has been used to isolate ligase–substrate complexes. However, there are several difficulties associated with this approach: Most ligase–substrate interactions are generally too weak and transient to isolate the substrates by immunoprecipitation, and the abundances of relevant in vivo substrates are often low due to proteasomal degradation. Recently, an antibody that recognizes the ubiquitin remnant motif Lys-e-Gly-Gly (diGly), which is exposed upon tryptic digestion 4630–4635 | PNAS | April 14, 2015 | vol. 112 | no. 15

The identification of specific ubiquitin ligase–substrate pairs is crucial for understanding the roles of protein ubiquitination in the regulation of diverse biological processes. Despite the development of various methodologies for substrate identification, it remains challenging to determine ubiquitin ligase substrates. Based on previously described tandem ubiquitinbinding entity(ies) (TUBE), we designed the trypsin-resistant (TR)-TUBE for expression in cells. The coexpression of TR-TUBE with an ubiquitin ligase stabilizes the ubiquitinated substrates by masking the ubiquitin chains. Using a combination of two strategies for enriching ubiquitinated substrates, TR-TUBE and anti–Lys-e-Gly-Gly antibody, we successfully identified specific ubiquitin ligase–substrate pairs. Our methodology provides an effective means for the identification of ubiquitin ligase substrates and the detection of ubiquitin ligase activity. Author contributions: Y.Y., Y.S., and K.T. designed research; Y.Y., Y.S., A.M., J.K., H.T., and H.Y. performed research; Y.Y., Y.S., H.T., H.Y., and M.S. analyzed data; and Y.Y., Y.S., and K.T. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

To whom correspondence may be addressed. Email: [email protected] or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1422313112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1422313112

-T

Ub Ub Ub Ub Substrate

TR

Ub Ub Ub Proteasome Ub Substrate

UB

E TR-TUBE IP: αFLAG WCL FLAG-TR-TUBE 24 48 72 posttransfection (h) 24 48 72 - + - + - + - + - + - + HA-Skp2 kDa 188 98 62 49 38 28 17 14

6 IP: αFLAG WCL FLAG-TR-TUBE 24 48 72 24 48 72 - + - + - + - + - + - +

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kDa 250 150 100 75 50 37

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- 4 8 Ubiquitin

100 80 60 40 20 0

conjugates

TR-TUBE mutant

0 24 48 72 posttransfection (h)

100 80 60 40 20 0

0 4 8 MG132 treatment (h)

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αUbiquitin

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IP: αFLAG Ub TR-TUBE FLAG-tagged HA-tagged emp p27 emp p27 - + - + - + - + HA-Skp2 - + - + - +- + - + -+ - + -+ MG132 kDa 250 150 100 75 50 37

(Ub)n -p27

25 20

25 20

6

C

Viability (%)

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DUB

48 emp

TR -T UB E

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WCL posttransfection (h) 0 24 48 72 TR-TUBE ++ + + TR-TUBE mutant + + + - - - - - - MG132 (h) kDa 188 IP: TR-TUBE 98 62 Lyse 49 38 Ub 28 Ub Ub 17 Ub 14 Substrate

B

1 2 3 4 5 6 7 8 9 10111213141516

αp27

αp27

TR-TUBE (Fig. 1A). We confirmed that the TR-TUBE can bind to all eight types of ubiquitin chain linkages (Fig. S1A). To examine the effect of overexpressed TR-TUBE on ubiquitin homeostasis and cytotoxicity, we first expressed TR-TUBE or an ubiquitin-binding–deficient TR-TUBE mutant (12) in 293T cells (Fig. S1 B and C). We then detected the cellular levels of free and conjugated ubiquitin by immunoblotting with an ubiquitinspecific antibody, and also analyzed cell death by propidium iodide staining (Fig. 1 B and C). The level of conjugated ubiquitin was increased by 48 h posttransfection, with a concomitant reduction in free ubiquitin (Fig. 1B). By contrast, we detected little accumulation of ubiquitin conjugates in cells expressing the ubiquitin-binding–deficient TR-TUBE mutant or in cells treated with the proteasome inhibitor MG132. The accumulation of ubiquitin conjugates upon TR-TUBE expression did result in some cell death at 48 h, comparable to the levels resulting from long-term treatment with MG132 (Fig. 1C). Although prolonged expression of TR-TUBE did not induce significant cell death, the accumulation of ubiquitin conjugates 72 h posttransfection in cells was reduced relative to the accumulation of ubiquitin conjugates at 48 h (Fig. 1 B and D), suggesting that cells highly expressing TR-TUBE gradually undergo cell death. To confirm that ubiquitinated substrates were actually included in the high-molecular-weight ubiquitin conjugates, we monitored the ubiquitination levels of p27/CDKN1B, one of the best-characterized ubiquitinated substrates. To this end, we expressed FLAG-tagged TR-TUBE or an ubiquitin-binding– deficient TR-TUBE mutant in cells with or without exogenously expressed Skp2/FBXL1, the F-box protein that recognizes p27 (13). Cell lysates were immunoprecipitated with an anti-FLAG Yoshida et al.

antibody, and both whole-cell lysates and precipitates were analyzed by immunoblotting (Fig. 1D and Fig. S2). Both ubiquitin conjugates and ubiquitinated p27 were efficiently enriched in the TR-TUBE immunoprecipitates. Although ubiquitin conjugates were detected in TR-TUBE mutant–expressing cells that were treated with proteasome/DUB inhibitors, ubiquitinated p27 was barely detectable, even when cells overexpressing Skp2 were treated with inhibitors (Fig. S2). By contrast, ubiquitinated p27 was detected in lysates of TR-TUBE– and Skp2overexpressing cells in the absence of inhibitor treatment, suggesting that TR-TUBE both protects the polyubiquitin chains on p27 from DUBs and circumvents proteasome degradation. As shown in Fig. 1D, ubiquitin conjugates and ubiquitinated p27 were detectable in TR-TUBE immunoprecipitates from cells harvested 24 h posttransfection in the absence of exogenous Skp2 expression. Notably, ubiquitinated p27 was present at high levels 48 h posttransfection in cells coexpressing Skp2 and TR-TUBE, suggesting that the ubiquitination of p27 detected in these samples was carried out by SCFSkp2, primarily exogenous Skp2. Detection of Ubiquitination Activity Using TR-TUBE. Conventionally, detection of specific ubiquitin ligase activity has been conducted using in vitro reconstitution or overexpression systems consisting of ubiquitin, a substrate, and an ubiquitin ligase in the presence of proteasome inhibitors. Indeed, we detected ubiquitination of p27 by SCFSkp2 in ubiquitin immunoprecipitates only when the substrate was overexpressed (Fig. 1E, lanes 5–8). However, overexpression of Skp2 failed to increase the ubiquitination of p27 to a detectable level. By contrast, in the presence of TR-TUBE, exogenous expression of Skp2 increased ubiquitination PNAS | April 14, 2015 | vol. 112 | no. 15 | 4631

BIOCHEMISTRY

Fig. 1. Protection of polyubiquitin chains on substrates by TR-TUBE. (A) TR-TUBE method for isolation of ubiquitinated substrates. Polyubiquitin chains on substrates are masked by exogenously expressed TR-TUBE, and thereby protected from DUBs and the proteasome. Ubiquitinated proteins are enriched by immunoprecipitation (IP) of TR-TUBE from cells expressing E3 ubiquitin ligase and TR-TUBE. Exogenously expressed proteins are shown in red. Ub, ubiquitin. (B) Accumulation of ubiquitin conjugates in TR-TUBE–expressing cells. 293T cells were transfected with FLAG-TR-TUBE or ubiquitin-binding–deficient FLAGTR-TUBE mutant plasmid, and the transfected cells were harvested at the indicated times. Cells transfected with HA-empty (emp) vector were treated with 10 μM MG132 for the indicated time before harvesting. Whole-cell lysates (WCLs) were analyzed by immunoblotting using antiubiquitin antibody. (C) Effect of TR-TUBE expression or MG132 treatment on cell viability, as determined by propidium iodide staining. Three independent plates of transfected or MG132 cells were analyzed. Error bars represent means ± SEM. (D) Detection of ubiquitin conjugates and ubiquitinated endogenous p27. Cells (1.3 × 106) were cotransfected with 3.5 μg of FLAG-TR-TUBE and 3.5 μg of HA-empty or HA-Skp2 expression plasmids, and the transfected cells were harvested at the indicated times. WCLs and anti-FLAG immunoprecipitates were analyzed by immunoblotting. The arrow indicates the position of p27. (E) Detection of ubiquitination of endogenous and overexpressed p27. Cells expressing FLAG-ubiquitin or FLAG-TR-TUBE with or without HA-Skp2 and/or HA-p27 were treated with or without MG132, and the cells were harvested at 48 h posttransfection. Anti-FLAG immunoprecipitates were analyzed by immunoblotting. The arrow indicates the position of p27.

of p27 (Fig. 1E, lanes 9–12): Ubiquitinated p27 was detectable even in the absence of exogenously overexpressed p27 or MG132. Because the elevated level of ubiquitin ligase was reflected by an increase in the accumulation of ubiquitinated substrates, the TR-TUBE system appears to be useful for quantitative detection of ubiquitin ligase activity toward a specific substrate. To suppress ubiquitination by endogenous Skp2, we next expressed the dominant-negative mutant Skp2ΔF, which lacks the F-box domain essential for binding to Cul1, thereby inhibiting formation of the SCF ubiquitin ligase complex. Although expression of the ΔF mutant failed to suppress the accumulation of ubiquitinated p27 in the presence of MG132, the absence of the inhibitor altered accumulation of ubiquitinated substrate but did not necessarily alter activity of the ligand (Fig. 2A). A similar ubiquitination pattern was detected for another Skp2 substrate, CDT1 (14). Next, we examined the ubiquitination activity of the other well-characterized F-box proteins, FBXW7 and FBXW1/ βTrCP1 (Fig. 2 B–D). The ubiquitination of c-Myc (15), a known substrate of FBXW7, was clearly increased by overexpression of FBXW7 and decreased by overexpression of its dominant-negative mutant (Fig. 2B). Overexpression of FBXW1 also increased the ubiquitination of NFKBIA (16) and PDCD4 (17), known FBXW1 substrates, even in the absence of stimuli (Fig. 2 C and D). Furthermore, we investigated the ubiquitination activity of RING-type E3 MDM2 for p53 (18) (Fig. 2E). The level of polyubiquitinated p53 was somewhat increased by overexpression of MDM2, whereas monoubiquitinated p53 was present at high levels, suggesting that MDM2 preferentially catalyzes monoubiquitination of p53 under these conditions (19). Furthermore, we examined the differences in the ubiquitination pattern of substrates due to overexpression of E3 in lysates prepared at different time points (Fig. S3). Although c-Myc was highly ubiquitinated 24 h posttransfection in cells expressing FBXW7, other ubiquitinated substrates accumulated at high levels 48 h posttransfection. Therefore, in subsequent experiments, we used lysates prepared from cells harvested 48 h posttransfection. Development of a Method to Identify Substrates for a Ubiquitin Ligase.

Recently, a large number of ubiquitination sites and ubiquitinated proteins were identified using the anti-diGly antibody (8, 9, 20). In those studies, this antibody was used for direct immunoprecipitation of trypsinized lysates prepared from cells treated with proteasome inhibitor. Therefore, to assess the efficiency of identification of ubiquitinated proteins using TR-TUBE, we compared three methods for enriching ubiquitinated peptides for liquid chromatography (LC)-tandem MS (MS/MS) analysis (Fig. 3A). These analyses were performed using starting material from ∼1 × 107 293T cells. The first and second methods used direct peptide immunoprecipitation, utilizing anti-diGly antibody, of trypsinized cell lysates from MG132-treated cells [diGly (MG132)] or TR-TUBE–overexpressing cells [diGly (FLAGTR-TUBE)]. In the direct peptide immunoprecipitation, cells were lysed in the presence of 9 M urea and the denatured proteins were diluted with Hepes buffer before trypsin digestion. By contrast, the third method used anti-FLAG antibody enrichment of TR-TUBE–associated proteins before peptide immunoprecipitation [FLAG and diGly (FLAG-TR-TUBE)]. Although the numbers of unique peptides containing the K-e-GG motif (diGly peptides) and ubiquitinated proteins did not differ significantly between these three methods, the ratios of diGly peptides to total identified peptides and ubiquitinated proteins were drastically different (Fig. 3B). In the dual-enrichment method, more than 95% of identified peptides included the K-e-GG motif. In addition, overexpression of TR-TUBE markedly decreased the abundance of identified peptides derived from ubiquitin, because TR-TUBE protected polyubiquitin chains on substrates from trypsin digestion (Fig. S4). These results show 4632 | www.pnas.org/cgi/doi/10.1073/pnas.1422313112

IP: αFLAG FLAG-TR-TUBE HA-tagged empSkp2 ΔF MG132 - + - + - + kDa 250 150 100 75 50 37

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IP: αFLAG IP: αFLAG FLAG-TR-TUBE FLAG-TR-TUBE empSkp2 ΔF HA-tagged emp W7 ΔF - + - + - + MG132 - +-+ -+ kDa kDa 250 250 150 150 100 100 75 75 50 50 37 37 25 25

B

αCDT1

αc-Myc

IP: αFLAG IP: αFLAG IP: αFLAG FLAG-TR-TUBE FLAG-TR-TUBE FLAG-TR-TUBE HA-tagged empW1 ΔF empW1 ΔF HA-tagged empW1 ΔF empW1 ΔF HA-tagged empMDM2 TNF - - - - - - + + + + + + Serum depl - - - - - - + + + + + + MG132 - + - + - + - + - + - + - + - + MG132 MG132 -+-+-+-+-+ -+

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Fig. 2. Detection of ubiquitination activity using TR-TUBE. Ubiquitination assays using the TR-TUBE method are shown. Cells (1.3 × 106) were cotransfected with 3.5 μg of plasmid encoding FLAG-TR-TUBE in combination with 3.5 μg of plasmid encoding emp, WT F-box protein [Skp2 (A), FBXW7 (W7; B), and FBXW1 (W1; C and D)], its dominant-negative mutant (ΔF), or MDM2 (E). Transfected cells were treated with or without MG132 for 4 h before harvesting. Cell lysates obtained 48 h posttransfection were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitates were analyzed by immunoblotting. Vertical bars and arrows denote the positions of ubiquitinated substrates and unmodified substrates, respectively.

that initial enrichment of ubiquitinated proteins using TR-TUBE reduces the background without loss of diGly peptides. To evaluate the performance of the TR-TUBE system in identification of ubiquitin ligase substrates, we next compared the number of peptide spectrum matches (PSMs) as a semiquantitative index of three known Skp2 substrates (p27, p21/CDKN1A, and CDT1), in mock-transfected cells, Skp2-expressing cells, and Skp2 ΔF-expressing cells (Fig. 3C). In the first method, using MG132-treated cells, p27 and p21 were barely detectable, whereas CDT1 was reproducibly observed. However, levels of the diGly peptides of these proteins were not always elevated in Skp2expressing cells. By contrast, in the methods using cells expressing TR-TUBE, especially in the dual-enrichment method, the levels of diGly peptides derived from the substrates were elevated in Skp2expressing cells. Although p27 itself was stabilized by MG132 treatment, ubiquitinated p27 was barely detectable in MG132treated cell lysates (Fig. 3D). By contrast, high-molecular-weight smears of both p27 and CDT1 were clearly detected in cells coexpressing TR-TUBE and WT Skp2. Notably, low levels of ubiquitinated CDT1 were detected in MG132-treated cells expressing either WT or mutant Skp2, but not in untreated cells, consistent with the results of the MS analyses. Thus, although there was little difference between the three methods with regard to the efficiency of identification of ubiquitinated proteins, enrichment of TR-TUBE–associated proteins before diGly peptide immunoprecipitation is an effective method for identifying substrates of an overexpressed ubiquitin ligase. In addition, we found that the levels of two ubiquitinated peptides derived from CKS1B were markedly elevated in Skp2expressing cells. CKS1B is an essential cofactor of SCFSkp2 that is necessary for the ubiquitination of p27 (21), and CKS1B itself is ubiquitinated by the APC/CCdh1 ubiquitin ligase (22). Hence, we investigated whether Skp2 ubiquitinates CKS1B by coexpressing myc-tagged CKS1B and TR-TUBE in cells, followed by immunoprecipitation of TR-TUBE and immunoblot analysis (Fig. 3E). Yoshida et al.

FLAG-TR-TUBE / empty

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Trypsin digestion

48 h post-transfection Lyse (urea lysis buffer)

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LC-MS/MS analysis WCL p27 p21 12 40 TR-TUBE empty 30 Skp2ΔF 8 HA-tagged Skp2 20 MG132 - + + + - - 4 10 0 0 kDa # experiment 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 250 diGly FLAG & diGly diGly diGly FLAG & diGly diGly (MG132) (TR-TUBE) (TR-TUBE) (MG132) (TR-TUBE) (TR-TUBE) 150 CKS1B 100 CDT1 16 60 75 empty Skp2ΔF 12 50 40 Skp2 37 8 20 4 25 20 0 0 # experiment 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 diGly diGly FLAG & diGly diGly diGly FLAG & diGly αp27 (MG132) (TR-TUBE) (TR-TUBE) (MG132) (TR-TUBE) (TR-TUBE) emp emp Skp2 ΔF emp Skp2 ΔF

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Fig. 3. Development of a method for identifying the substrates of an ubiquitin ligase. (A) Schematic indicating the steps in the substrate identification processes. In the diGly (MG132) method, 293T cells were transfected with HA-empty (empty), ΔF, or WT F-box protein, and the transfectants were treated with MG132 for 4 h before harvesting. In the diGly (TR-TUBE) and FLAG and diGly (TR-TUBE) methods, cells were transfected with FLAG-TR-TUBE in combination with HA-empty, ΔF, or WT F-box protein. In the diGly (MG132) and diGly (TR-TUBE) methods, cells were lysed 48 h posttransfection in urea-based solution and diluted WCLs were digested with trypsin. In the FLAG and diGly (TR-TUBE) method, cells were lysed and immunoprecipitated with anti-FLAG antibody and the eluted proteins from immunoprecipitates were digested with trypsin. The tryptic peptides were further enriched in another immunoaffinity step for peptides containing the K-e-GG motif (peptide IP with anti-diGly antibody), followed by MS analysis. (B) Comparison of the numbers of diGly peptide (Upper) and ubiquitinated protein (Lower) numbers identified by the three methods described in A. Five individual experiments were performed for each method. (C) Comparison of the efficiency of identification of Skp2 substrates by the three methods described in A. Total peptide spectrum match numbers (# PSMs) of the indicated proteins (p27, p21, CDT1, and CKS1B) obtained from LC-MS/MS analysis using cells expressing HA-empty (blue bars), dominantnegative mutant (Skp2ΔF, green bars), or WT Skp2 (red bars) with or without FLAG-TR-TUBE are shown. Three individual experiments were performed. (D) Detection of ubiquitinated p27 and CDT1 in MG132-treated cells and TR-TUBE–expressing cells. Vertical bars indicate ubiquitinated substrates, and arrows indicate the positions of unmodified substrates. (E) Testing the ubiquitination of CKS1B by Skp2. The arrow indicates the position of 6× Myc-CKS1B.

As predicted, the expression of Skp2 stimulated the ubiquitination of CKS1B. The identified ubiquitination sites of Skp2 substrates are listed in Table S1, all of which were included in the PhosphoSite Plus and neXtProt databases. Identification of Substrates for Uncharacterized Ubiquitin Ligases.

Our next goal was to develop a method for the systematic identification of ubiquitin ligase–substrate pairs. For this purpose, we examined the F-box proteins constituting the SCF complex in 293T cells and identified 12 F-box proteins from a FLAG-Cul1 immunoprecipitate (Table S2). We attempted to find substrates for FBXO21, which is ubiquitously expressed and has not yet been well characterized. To screen for substrates of FBXO21, we performed LC-MS/MS analysis of peptides prepared by the dual-enrichment method from cells coexpressing FLAG-TR-TUBE and HA-empty, HA-FBXO21ΔF, or HA-FBXO21. In three independent analyses, we selected substrate candidates whose PSM numbers and protein scores increased in FBXO21-expressing cells but decreased in FBXO21ΔF-expressing cells (Table S3). Of these candidates, we picked threonyl-tRNA synthetase (TARS) and EP300 interacting inhibitor of differentiation 1 (EID1) because of their reproducibility (Fig. 4A). We also confirmed that the interaction of FBXO21 with TARS or EID1 was stabilized by Skp1 coexpression (23) and treatment with the Nedd8 E1 enzyme inhibitor Yoshida et al.

MLN4924, which stabilizes Cullin-RING ligase substrates (20) (Fig. S5A). We cloned several F-box proteins and investigated whether they bound EID1 or TARS in the presence of MLN4924 (Fig. S5B). Although TARS was detectable at low levels in a few F-box protein immunoprecipitates, the levels of TARS and EID1 were most prominent in FBXO21 immunoprecipitate. EID1, first cloned as an RB1-binding protein, has the ability to inhibit p300 (24) and can also interact with the orphan nuclear receptor SHP (25). To determine whether the interaction of EID1 with FBXO21 is mediated by RB1 or SHP, we sought to identify the regions on EID1 that are necessary for FBXO21 interaction (Fig. S5C). Deletion of the C-terminal RB1-binding site of EID1 did not affect the binding to FBXO21, but deletion of an additional nine residues abolished interaction, suggesting that their interaction is independent on RB1 and SHP. Next, we used immunoblot analysis to evaluate the ability of FBXO21 to ubiquitinate EID1. Ubiquitinated EID1 was clearly detected in both FBXO21-expressing and MG132-treated cells (Fig. 4B), and knockdown of FBXO21 by siRNA suppressed accumulation of ubiquitinated EID1 in MG132-treated cells (Fig. 4C). Further, ubiquitination of EID1 was clearly increased by overexpression of FBXO21, but not other the F-box proteins tested (Fig. 4D). As shown in Fig. 4E, knockdown of FBXO21 by siRNA stabilized EID1 protein levels but did not affect EID1 mRNA levels (Fig. 4F). Consistent with the steady-state levels of EID1, FBXO21 knockdown, as well as treatment of cells with MLN4924, led PNAS | April 14, 2015 | vol. 112 | no. 15 | 4633

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48 h post-transfection Lyse (urea lysis buffer)

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emp emp Skp2 ΔF emp Skp2 ΔF

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FB X FB W1 X FB W7 FBXW8 X Sk W11 p FB 2 X em L12 p FB ty XO FB 5 X FB O7 FBXO9 X FB O11 X FB O21 X FB O22 XO 44

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co n FB t X Rb O21 MD1 co M2 n FB t X Rb O21 MD1 M2

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50 37 25

αEID1

Fig. 4. Identification of substrates for FBXO21. (A) Screening of FBXO21 substrates by the dual-enrichment method using TR-TUBE and anti-diGly antibody. In three sets of independent MS analyses, we selected proteins whose PSM numbers (# PSMs) increased in cells expressing FBXO21 and decreased in cells expressing FBXO21 mutant. The total PSM numbers of identified substrates (TARS and EID1) obtained from LC-MS/MS analysis using cells expressing HA-empty (blue bars), ΔF (green bars), or WT FBXO21 (red bars) with FLAG-TR-TUBE are shown. (B and C) Ubiquitination assay of EID1. Forty-four hours after FLAG-TR-TUBE in combination with emp, WT FBXO21 (F21), its mutant (ΔF), or siRNA transfection, cells were treated with or without MG132 for 4 h. AntiFLAG immunoprecipitates were analyzed by immunoblotting. Arrows show the positions of EID1. cont, control. (D) Ubiquitination assay of EID1 by using TR-TUBE and various F-box proteins. Cells were transfected with plasmids encoding HA-TR-TUBE and each FLAG-tagged F-box protein. Anti-HA immunoprecipitates were analyzed by immunoblotting. The arrow shows the position of EID1. (E) RNAi-mediated knockdown of FBXO21. Forty-eight hours after siRNA (si) transfection, FBXO21 and EID1 protein levels in WCLs were assessed by immunoblotting. (F) Quantitative RT-PCR analysis. Total RNA was prepared from 293T cells 48 h after cells were transfected with control or FBXO21-specific siRNA. The data shown are representative of three independent experiments. (G) Forty-eight hours after siRNA transfection, cells were incubated with 2 μg/mL cycloheximide (CHX). In parallel, cells were treated with 1 μM MLN4924 for 1 h before addition of cycloheximide. Cells were harvested at the indicated times after cycloheximide treatment, and WCLs were analyzed by immunoblotting. (H) Stabilization of EID1 by depletion of FBXO21. Twenty-four hours or 48 h after transfection of the indicated siRNAs, protein levels in WCL were analyzed by immunoblotting. (I) Ubiquitination of EID1 by MDM2 and F21. The arrow shows the position of EID1.

to stabilization of EID1 in a cycloheximide chase experiment, extending the t1/2 from 3 h (Fig. 4G). These results indicate that EID1 is degraded by the proteasome following ubiquitination by SCFFBXO21. MDM2, rather than Cullin-RING ubiquitin ligase, is the ubiquitin ligase for EID1 (26, 27). However, siRNA-mediated knockdown of MDM2 or RB1 did not stabilize EID1 protein in 293T cells (Fig. 4H). RB1 was stabilized by knockdown of MDM2 at 24 h, consistent with the fact that MDM2 promotes degradation of RB1 (28, 29). In addition, interaction with EID1 causes a conformational rearrangement of RB1 (29, 30), and their interaction may cause RB1 stabilization because the RB1 level was also increased by knockdown of FBXO21 (Fig. 4H). Furthermore, we examined the ubiquitination activity of MDM2 for EID1 (Fig. 4I). However, ubiquitinated EID1 was barely detectable in MDM2-expressing cells, suggesting that the stability of EID1 is regulated by SCFFBXO21 under normal conditions. TARS is an aminoacyl-tRNA synthetase that is as abundant in cells as ribosomal proteins (31). Although TARS is a highly abundant and stable protein due to its fundamental roles in protein synthesis, 13 residues in TARS are ubiquitinated by Cullin-RING ubiquitin ligases (9), and we found that four sites were ubiquitinated by SCFFBXO21 (Table S1). Indeed, ubiquitination of TARS was stimulated by overexpressed FBXO21 (Fig. S6 A and B). However, the levels of endogenous TARS protein remained constant regardless of whether FBXO21 was knocked down (Fig. S6 C–E). Furthermore, we showed that FBXO21 interacted 4634 | www.pnas.org/cgi/doi/10.1073/pnas.1422313112

with the TARS editing domain, which includes all of the identified ubiquitination sites within the TARS protein (Fig. S6F and Table S1). In Escherichia coli, severe oxidative stress reduced overall translational fidelity by impairing the editing activity of TARS (24). Actually, ubiquitination of TARS by SCFFBXO21 was slightly elevated in H2O2-treated cells (Fig. S6G), but the stress did not affect its stability (Fig. S6H). These results suggest that TARS is regulated by SCFFBXO21 ubiquitination under stress conditions but that its degradation is not detectable because the protein is so abundant. Editing-defective aminoacyl-tRNA synthetase causes protein misfolding and neurodegeneration (32), and stresses other than oxidative stress may damage its editing activity. Further studies are needed to determine how FBXO21 detects damage in the TARS editing domain. Discussion In this study, we developed the TR-TUBE system, which is useful for detecting ubiquitin ligase activity and identifying substrates of specific E3 ubiquitin ligases. In this system, overexpressed E3 ubiquitinates its endogenous substrates by using ubiquitination-related factors present in cells, and TR-TUBE prevents degradation and deubiquitination of these substrates, allowing detection of the specific activity of an E3 and isolation of its substrates. Although TR-TUBE immunoprecipitates contain excess ubiquitin, TR-TUBE can protect ubiquitin chains on substrates from trypsin digestion (Fig. S4) and reduce the proportion of peptides derived from ubiquitin, which hinder identification of Yoshida et al.

1. Grabbe C, Husnjak K, Dikic I (2011) The spatial and temporal organization of ubiquitin networks. Nat Rev Mol Cell Biol 12(5):295–307. 2. Komander D, Rape M (2012) The ubiquitin code. Annu Rev Biochem 81:203–229. 3. Deshaies RJ, Joazeiro CA (2009) RING domain E3 ubiquitin ligases. Annu Rev Biochem 78:399–434. 4. Jin J, et al. (2004) Systematic analysis and nomenclature of mammalian F-box proteins. Genes Dev 18(21):2573–2580. 5. Busino L, et al. (2007) SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins. Science 316(5826):900–904. 6. Davis MA, et al. (2013) The SCF-Fbw7 ubiquitin ligase degrades MED13 and MED13L and regulates CDK8 module association with Mediator. Genes Dev 27(2):151–156. 7. Tan MK, Lim HJ, Harper JW (2011) SCF(FBXO22) regulates histone H3 lysine 9 and 36 methylation levels by targeting histone demethylase KDM4A for ubiquitin-mediated proteasomal degradation. Mol Cell Biol 31(18):3687–3699. 8. Xu G, Paige JS, Jaffrey SR (2010) Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol 28(8):868–873. 9. Kim W, et al. (2011) Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 44(2):325–340. 10. Sarraf SA, et al. (2013) Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496(7445):372–376. 11. Hjerpe R, et al. (2009) Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep 10(11):1250–1258. 12. Sasaki T, Funakoshi M, Endicott JA, Kobayashi H (2005) Budding yeast Dsk2 protein forms a homodimer via its C-terminal UBA domain. Biochem Biophys Res Commun 336(2):530–535. 13. Carrano AC, Eytan E, Hershko A, Pagano M (1999) SKP2 is required for ubiquitinmediated degradation of the CDK inhibitor p27. Nat Cell Biol 1(4):193–199. 14. Li X, Zhao Q, Liao R, Sun P, Wu X (2003) The SCF(Skp2) ubiquitin ligase complex interacts with the human replication licensing factor Cdt1 and regulates Cdt1 degradation. J Biol Chem 278(33):30854–30858. 15. Yada M, et al. (2004) Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7. EMBO J 23(10):2116–2125. 16. Tan P, et al. (1999) Recruitment of a ROC1-CUL1 ubiquitin ligase by Skp1 and HOS to catalyze the ubiquitination of I kappa B alpha. Mol Cell 3(4):527–533. 17. Dorrello NV, et al. (2006) S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science 314(5798):467–471. 18. Hock AK, Vousden KH (2014) The role of ubiquitin modification in the regulation of p53. Biochim Biophys Acta 1843(1):137–149. 19. Li M, et al. (2003) Mono- versus polyubiquitination: Differential control of p53 fate by Mdm2. Science 302(5652):1972–1975.

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substrates of ubiquitin ligases, it requires parameters such as linker length and configuration to be optimized for each F-box protein. By contrast, the TR-TUBE strategy is very simple, requiring only coexpression of TR-TUBE and the E3. Furthermore, the TR-TUBE method is not restricted to SCF-type ubiquitin ligases, and is also potentially useful for other E3 families. By using a combination of two enrichment strategies, TR-TUBE and the anti–K-e-GG antibody, we succeeded in identifying ubiquitinated substrates from small amounts of cell lysate. Thus, the TRTUBE system represents a practical means for obtaining important insights into the functions of ubiquitin ligases. Materials and Methods For immunoaffinity purification for ubiquitinated protein identification, WCL prepared from a 10-cm cell culture dish harvested 48 h posttransfection (∼1 × 107 cells) was incubated for 1 h with anti-FLAG monoclonal antibody (antiDDDDK)–conjugated agarose beads (MBL International). Bead-bound proteins were eluted FLAG peptide (Sigma). Proteins were reduced in 5 mM Tris [2-carboxy-ethyl] phosphine hydrochloride for 30 min at 50 °C, and then alkylated with 10 mM methylmethanethiosulfonate, and alkylated proteins were digested overnight at 37 °C with 1 μg of trypsin (Promega). After tryptic digestion, ubiquitinated peptides were enriched by using the PTMScan ubiquitin remnant motif (K-e-GG) kit (Cell Signaling). The eluted peptides were desalted using GL-Tip SDB and GL-Tip GC (GL Sciences) prior to LC-MS analysis. Detailed methods are provided in SI Materials and Methods. ACKNOWLEDGMENTS. This work was supported by a Grant-in-Aid [Japan Society for the Promotion of Science (JSPS) KAKENHI Grant 24580152] for Scientific Research on a Priority Area (to Y.Y.), a Grant-in-Aid (JSPS KAKENHI Grant 24112008) for Scientific Research on Innovative Areas (to Y.S.), Grantsin-Aid (JSPS KAKENHI Grants 2611377 and 13J07852) for JSPS Fellows (to H.T. and H.Y., respectively), and a Grant-in-Aid (JSPS KAKENHI Grant 21000012) for Specially Promoted Research (to K.T.).

20. Emanuele MJ, et al. (2011) Global identification of modular cullin-RING ligase substrates. Cell 147(2):459–474. 21. Ganoth D, et al. (2001) The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27. Nat Cell Biol 3(3):321–324. 22. Bashir T, Dorrello NV, Amador V, Guardavaccaro D, Pagano M (2004) Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase. Nature 428(6979):190–193. 23. Yoshida Y, Murakami A, Tanaka K (2011) Skp1 stabilizes the conformation of F-box proteins. Biochem Biophys Res Commun 410(1):24–28. 24. Ling J, Söll D (2010) Severe oxidative stress induces protein mistranslation through impairment of an aminoacyl-tRNA synthetase editing site. Proc Natl Acad Sci USA 107(9):4028–4033. 25. Båvner A, Johansson L, Toresson G, Gustafsson JA, Treuter E (2002) A transcriptional inhibitor targeted by the atypical orphan nuclear receptor SHP. EMBO Rep 3(5): 478–484. 26. Miyake S, et al. (2000) Cells degrade a novel inhibitor of differentiation with E1A-like properties upon exiting the cell cycle. Mol Cell Biol 20(23):8889–8902. 27. Ye B, et al. (2014) Pcid2 inactivates developmental genes in human and mouse embryonic stem cells to sustain their pluripotency by modulation of EID1 stability. Stem Cells 32(3):623–635. 28. Uchida C, et al. (2005) Enhanced Mdm2 activity inhibits pRB function via ubiquitindependent degradation. EMBO J 24(1):160–169. 29. Sdek P, et al. (2005) MDM2 promotes proteasome-dependent ubiquitin-independent degradation of retinoblastoma protein. Mol Cell 20(5):699–708. 30. Hassler M, et al. (2007) Crystal structure of the retinoblastoma protein N domain provides insight into tumor suppression, ligand interaction, and holoprotein architecture. Mol Cell 28(3):371–385. 31. Kulak NA, Pichler G, Paron I, Nagaraj N, Mann M (2014) Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods 11(3):319–324. 32. Lee JW, et al. (2006) Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration. Nature 443(7107):50–55. 33. Jin J, Arias EE, Chen J, Harper JW, Walter JC (2006) A family of diverse Cul4-Ddb1interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Mol Cell 23(5):709–721. 34. Shirane M, et al. (1999) Down-regulation of p27(Kip1) by two mechanisms, ubiquitinmediated degradation and proteolytic processing. J Biol Chem 274(20):13886–13893. 35. Mark KG, Simonetta M, Maiolica A, Seller CA, Toczyski DP (2014) Ubiquitin ligase trapping identifies an SCF(Saf1) pathway targeting unprocessed vacuolar/lysosomal proteins. Mol Cell 53(1):148–161.

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substrates in LC-MS/MS analysis. In trypsinized TR-TUBE immunoprecipitates, substrates of coexpressed Skp2 could be detected by LC-MS/MS analysis; however, we also detected other proteins that are abundant in complex with these substrates, as well as Skp2 itself, but no diGly peptides other than those diGly peptides derived from ubiquitin and Skp2. Therefore, the second enrichment with antidiGly antibody is remarkably effective for identification of substrates of a particular ubiquitin ligase. All of the ubiquitination sites of substrates identified in this study were included among the known ubiquitination sites contained in databases (Table S1). However, some sites identified in CDT1 and TARS differed from the sites reported to be ubiquitinated by the Cullin-RING ubiquitin ligase (9). In addition to three reported ubiquitination sites (K132, K153, and K165) in CDT1, which our system failed to detect, K24 of CDT1 was ubiquitinated by SCFSkp2. CDT1 is ubiquitinated by both SCFSkp2 (14) and Cul4-Ddb1 (33), suggesting that the reported sites are ubiquitinated by Cul4-Ddb1. Notably, although the ubiquitination sites of p27 have been analyzed by site-directed mutagenesis (34), these sites have not previously been assigned by proteomic analyses. The diGly peptides derived from p27 were barely detectable in peptide immunoprecipitates of trypsinized cell lysates from cells treated with proteasome inhibitor, but they were effectively detected in lysates from TR-TUBE– expressing cells, suggesting that deubiquitination of p27, rather than instability of the protein itself, hampers detection of the diGly peptides. Recently, the ubiquitin ligase substrate trapping method was developed for the isolation of ubiquitinated substrates in yeast. In that method, ligase–substrate affinity is increased by fusing the ligase to a tandem ubiquitin-associated domain (35). Although this method should also be useful for identification of the

Supporting Information Yoshida et al. 10.1073/pnas.1422313112 SI Materials and Methods Plasmid Construction. To construct a polyubiquitin-binding probe, we modified a previously reported high-affinity probe for polyubiquitin, TUBE (1). cDNA encoding the ubiquitin-associated (UBA) domain of human UBQLN1 (NM_013438) was subcloned into a pBSKS (+) vector, and three Arg residues were substituted with Ala residues using a QuikChange Site-Directed Mutagenesis II Kit (Agilent). DNA encoding four tandem copies of the UBA domains, joined by the flexible linker sequence (GlyGlyGlySerGlyGlyGly), was subcloned into vector pcDNA3FLAG. The coding region sequence of the resulting plasmid, called “FLAG-TR-TUBE,” is shown in Fig. S1B. The L368A/ L369A mutant abolishes ubiquitin binding by the UBA domain of Dsk1 (2). To construct an ubiquitin-binding–deficient TR-TUBE mutant, we generated a synthetic gene encoding the TR-TUBE mutant in which two conserved Leu residues, corresponding to L368L369 of Dsk1, were replaced with Ala residues in four positions simultaneously, and we then subcloned the resultant construct into pcDNA3-FLAG (Fig. S1C). cDNA encoding Skp2 (NM_013787), FBXW7 (NM_033632), FBXW1/ BTRC (NM_003939), FBXO21 (NM_015002), the ΔF mutants of the preceding genes, MDM2 (XM_005268872), or CDKN1B (NM_004064) was subcloned into pcDNA3-HA. cDNA encoding CKS1B (NM_001826) or TARS (NM_15295) was subcloned into pcDNA3.1-6× Myc. For FLAG-tagged F-box protein expression, cDNA encoding FBXW1/BTRC, FBXW7, FBXW8 (NM_153348); FBXW11 (NM_033645); Skp2, FBXL12 (NM_017703); FBXO5 (NM_012177); FBXO7 (NM_012179); FBXO9 (NM_012347); FBXO11 (NM_001190274); FBXO21, FBXO22 (NM_147188); or FBXO44 (NM_183412) was subcloned into pcDNA3-FLAG. Cell Culture and Transfection. 293T cells (1.3 × 106) were cultured in DMEM supplemented with 10% (vol/vol) FBS in a 10-cm cell culture dish for 24 h. Cells were transiently transfected with 3.5 μg of FLAG-tagged expression plasmid (TR-TUBE or ubiquitin) and 3.5 μg of HA-tagged F-box protein plasmid with 21 μg of polyethylenimine (Polysciences). Cell Viability Assay. 293T cells (1 × 105) were cultured in a six-well cell culture plate for 24 h. Cells were transiently transfected with 2 μg of plasmid. Harvested cells were suspended in cell culture medium, and propidium iodide was added at a final concentration of 2 μg/mL. After incubation for 5 min, the numbers of stained or total cells were counted in “viability” mode on a Tali Image-Based Cytometer (Life Technologies). In Vitro Diubiquitin-Binding Assay. cDNA encoding TR-TUBE was

cloned into pRSET-A (Life Technologies), in which a Ser residue upstream of 6× His tag was substituted with a Cys residue to permit biotinylation. The resultant His-TR-TUBE was expressed in E. coli Rosetta2 (DE3) for 15 h at 22 °C. Bacterial cells were lysed by passage through a precooled French pressure cell (Ohtake Works) in lysis buffer [50 mM sodium phosphate, 300 mM NaCl, 10% glycerol, 1 mM Tris [2-carboxyethyl] phosphine hydrochloride (pH 7.0), 1 mM Tris [2-carboxy-ethyl] phosphine hydrochloride (TCEP)], and the lysate was clarified by centrifugation. The supernatant was incubated with TALON resin (Clontech), and TR-TUBE was eluted with elution buffer [50 mM sodium-Hepes (pH 7.1), 100 mM NaCl, 0.2 M imidazole]. Then, TR-TUBE was biotinylated with EZ-link Maleimide-PEG2-Biotin (Thermo Scientific) and further purified by gel filtration on Superdex 75 Yoshida et al. www.pnas.org/cgi/content/short/1422313112

10/100 GL (GE Healthcare) preequilibrated with 50 mM Hepes (pH 7.5), 100 mM NaCl, and 10% glycerol. To conjugate His-TR-TUBE to beads, 2 μg of biotinylated HisTR-TUBE was incubated with a 50-μL suspension of Dynabeads MyOne Streptavidin C1 (Life Technologies) for 30 min, and the beads were washed twice. The conjugated TR-TUBE was incubated with 2 μg of each diubiquitin (M1, K6, K11, K27, K29, K33, K48, or K63; Boston Biochem) in 20 μL of 0.1% TritonX100 in 50 mM ammonium bicarbonate for 30 min. After incubation, the supernatant (unbound fraction) was removed and the beads were washed three times with the incubation buffer. The bound diubiquitin and TR-TUBE (bound fraction) were eluted by boiling with SDS sample buffer. Both unbound and bound fractions were subjected to SDS/PAGE and stained with Coomassie Brilliant Blue. Immunoprecipitation and Immunoblotting. To inhibit cellular proteasome activity, cells were treated with 10 μM MG132 (Peptide Institute, Inc.) 4 h before harvesting. For TNF-α stimulation, cells were treated with 3 μg/mL TNF-α for 20 min before harvesting. To inhibit NEDD8-activating enzyme activity, cells were treated with 1 μM MLN4924 (Boston Biochem) 14 h before harvesting. Forty-eight hours after transfection, cells were lysed with TBS-N [10 mM Tris·HCl (pH 7.5), 150 mM NaCl, 0.5% Nonidet P-40] containing protease inhibitor mixture (cOmplete, EDTA-free; Roche). For inhibition of DUB activity, cells were lysed with TBS-N in the presence of 1 mM N-ethylmaleimide. Lysates were centrifuged at 20,000 × g for 20 min at 4 °C. The supernatants [whole-cell lysate (WCL)] were immunoprecipitated with anti-FLAG monoclonal antibody (anti-DDDDK)-conjugated agarose (MBL International) or anti-HA monoclonal antibody (HA-7)-conjugated agarose (Sigma–Aldrich) for 1 h at 4 °C. Immunoprecipitates and cell lysates were subjected to immunoblot analysis as previously described, with antibodies against ubiquitin (1:500 dilution, clone P4D1; Santa Cruz Biotechnology), p27 (1:1,000, Rabbit anti-p27 Kip1 D69C12; Cell Signaling,), CDT1 (1:1,000, Rabbit D10F11; Cell Signaling), NFKBIA (1:1,000, Rabbit 44D4; Cell Signaling,), PDCD4 (1:1,000, Rabbit D29C6; Cell Signaling), p53 (1:1,000, Rabbit 7F5; Cell Signaling), c-Myc [1:200, Rabbit N-262; Santa Cruz Biotechnology (for Myc-tag: 1:1,000, Rabbit; Cell Signaling)], TARS (1:200, Rabbit H-100; Santa Cruz Biotechnology), EID1 (1:500, Rabbit; Proteintech), FBXO21 (1:500, Rabbit; GeneTex), Rb1 (1:2,000, clone 4H1; Cell Signaling), and MDM2 (1:200, clone SMP14; Santa Cruz Biotechnology). Immunoaffinity Purification and Trypsin Digestion for Ubiquitinated Protein Identification. All steps were performed at 4 °C unless

otherwise noted. For FLAG-TR-TUBE immunoprecipitation, 2 mL of WCL (∼1.5 mg/mL) prepared from a 10-cm cell culture dish harvested 48 h posttransfection (∼1 × 107 cells) was incubated for 1 h with 25 μL of anti-FLAG monoclonal antibodyconjugated agarose beads. After the agarose beads were washed five times with 1.5 mL of TBS-N and twice with 1.5 mL of 50 mM ammonium bicarbonate (AMBIC), bead-bound proteins were eluted three times with 25 μL of 200 μg/mL FLAG peptide (Sigma) in 50 mM AMBIC. Proteins were reduced in 5 mM TCEP for 30 min at 50 °C and then alkylated with 10 mM methylmethanethiosulfonate (MMTS) for 10 min at room temperature. Next, alkylated proteins were digested overnight at 37 °C with 1 μg of trypsin (Promega). After tryptic digestion, samples were acidified to ∼pH 2 with TFA and desalted by 1 of 11

solid-phase extraction using GL-Tip SDB and GL-Tip GC (both from GL Sciences). For direct immunoprecipitation of ubiquitinated peptides, ∼1 × 107 harvested cells were washed with PBS and then lysed by sonication in urea lysis buffer [20 mM Hepes (pH 8), 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate]. Lysates were centrifuged at 20,000 × g for 15 min at room temperature. Proteins in the supernatants were reduced in 5 mM TCEP for 2 h at 37 °C and alkylated with 10 mM MMTS for 10 min at room temperature. The resultant solution was diluted with 3 vol of 20 mM Hepes (pH 8), and proteins were digested overnight with 10 μg of trypsin at 37 °C with rotation. After tryptic digestion, samples were acidified to ∼pH 2 with TFA and centrifuged at 20,000 × g for 15 min; the resultant supernatant was applied to a Sep-PaK Light C18 cartridge (WAT023501; Waters) for peptide purification. Immunoprecipitation of diGly-Containing Peptide. Ubiquitinated peptides were enriched by using the PTMScan Ubiquitin Remnant Motif (K-e-GG) Kit (Cell Signaling). Briefly, eluted peptides were dried by vacuum centrifugation, dissolved in 0.2 mL of IAP buffer [50 mM Mops (pH 7.2), 10 mM Na2HPO4, 50 mM NaCl], adjusted to pH 7 with 1 M Tris, and incubated for 2 h at 4 °C with 10 μL of ubiquitin branch motif immunoaffinity beads. Then, the beads were washed twice with 250 μL of IAP buffer and three times with 250 μL of distilled water, and peptides were eluted with 3 × 20 μL of 0.15% TFA. The eluted peptides were desalted using GL-Tip SDB and GL-Tip GC before LC-MS analysis. Immunoaffinity Purification and Trypsin Digestion for Cul1- or F-Box Protein–Associated Protein Identification. Immunoprecipitations

were performed by incubating WCL (∼1.5 mg/mL) with antiFLAG monoclonal antibody and Dynabeads Protein G (Life Technologies) for 1 h. After the Dynabeads were washed four times with 200 μL of TBS-N, immune complexes were eluted three times with 20 μL of FLAG peptide (200 μg/mL; Sigma) and boiled for 5 min in 50 mM AMBIC with 0.1% Rapigest (Millipore). Next, denatured proteins were alkylated with MMTS and digested overnight at 37 °C with 1 μg of trypsin. After tryptic digestion, samples were incubated in 0.5% TFA at 37 °C for 1 h and then centrifuged at 20,000 × g for 15 min to remove Rapigest. The resultant supernatant was applied to GL-Tip SDB and GL-Tip GC. MS Analysis. Desalted tryptic digests were analyzed by nanoflow ultra-HPLC (Easy nLC; Thermo Scientific) coupled to a Q Exactive mass spectrometer (Thermo Scientific). The mobile phases were 0.1% formic acid in water (solvent A) and 0.1% formic acid in 100% acetonitrile (solvent B). Peptides were directly loaded onto a C18 analytical column [ReproSil-Pur (3 μm, 75-μm i.d., and 12-cm length); Nikkyo Technos] and separated using a 150-min two-step gradient (0–40% in 120 min, 40–100% in 20 min, and 100% in 10 min of solvent B) at a constant flow rate of 300 nL·min−1. For ionization, 1.8 kV of liquid junction voltage and a capillary temperature of 250 °C were used. The Q Exactive mass spectrometer was operated in the data-dependent MS/MS mode, using Xcalibur software (Thermo Scientific), with survey scans acquired at a resolution of 70,000 at m/z 200. The top 10 most abundant isotope patterns with a charge ranging 1. Hjerpe R, et al. (2009) Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep 10(11):1250–1258.

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from 2–4 were selected from the survey scans with an isolation window of 2.0 m/z and fragmented by higher-energy collisional dissociation with normalized collision energies of 28. The maximum ion injection times for the survey and MS/MS scans were 60 ms, and the ion target values were set to 3e6 and 1e5 for the survey and MS/MS scans, respectively. Ions selected for MS/MS were dynamically excluded for 5 s for diGly peptide identification or for 90 s for binding protein identification. Protein Identification from MS Data. Proteome Discoverer software (version 1.3; Thermo Scientific) was used to generate peak lists. The MS/MS spectra were searched against a UniProt Knowledgebase (version 2012_10) using the Mascot search engine. The precursor and fragment mass tolerances were set to 10 ppm and 20 millimass units, respectively. Methionine oxidation, protein amino-terminal acetylation, pyroglutamate formation, Ser/Thr/ Tyr phosphorylation, and diglycine modification of Lys side chains were set as variable modifications, and Cys methylthio modification was set as a static modification for database searching. Peptide identification was filtered at a 1% false discovery rate. To identify specific substrates of ubiquitin ligases, the results of three individual samples (cells expressing FLAG-TR-TUBE with empty vector, WT ubiquitin ligase, or dominant-negative mutant ubiquitin ligase) were assembled into one multiconsensus report using Proteome Discoverer software. Cumulative protein scores were compared based on summing the ion scores and the total numbers of identified sequences (PSMs) of ubiquitinated peptides (peptides containing Lyse-Gly-Gly). RNAi Experiment. SMART pool: siGENOME FBXO21, Rb1, and MDM2 siRNAs and a nonspecific control duplex were purchased from Dharmacon. Transfection of siRNAs into cells was accomplished using Lipofectamine 2000 (Invitrogen) at a final concentration of 50 nM in six-well dishes. Quantitative Real-Time PCR Assay. The cDNA was synthesized from 1 μg of total RNA using a Transcriptor First Strand cDNA Synthesis Kit (Roche). Quantitative PCR was performed using LightCycler 480 Probes Master (Roche) in a LightCycler 480 (Roche). Signals were normalized to the glyceraldehyde-3phosphate dehydrogenase gene (GAPDH). The forward and reverse primer pairs were 5′-GCAATGTACCCGGACCAG-3′ and 5′-TGTCAAGCACCTTCAAAACAA-3′ for FBXO21, 5′-AACGGAGCCTTGCTAACG-3′ and 5′-TCCTCGCTCTCGAAGTCTG-3′ for EID1, 5′-ATATCAAATTGCCTGTGGAATTAGT-3′ and 5′-TCCAAGGTACAATCTTCTTCCAG-3′ for TARS, and 5′-AGCCACATCGCTCAGACAC-3′ and 5′-GCCCAATACGACCAAATCC-3′ for GAPDH. Cycloheximide Chase Assay. 293T cells (1 × 105) in 6-cm cell culture dishes were transfected with siRNA. The cells were then cultured for 48 h, followed by incubation with 2 μg/mL cycloheximide. For MLN4924 treatment, cells were treated with 1 μM MLN4924 for 1 h before cycloheximide addition. For oxidative stress, cells were incubated with 2 mM H2O2 for 30 min, washed twice with medium, and then incubated with cycloheximide. After incubation for the indicated periods, the cells were washed with PBS and suspended in TBS-N containing protease inhibitor mixture. Lysates (30 μg) were subjected to immunoblot analyses. 2. Sasaki T, Funakoshi M, Endicott JA, Kobayashi H (2005) Budding yeast Dsk2 protein forms a homodimer via its C-terminal UBA domain. Biochem Biophys Res Commun 336(2):530–535.

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Fig. S1. Affinity of TR-TUBE for ubiquitin chain linkages and sequence of TR-TUBE. (A) In vitro diubiquitin (Ub2 or Ub2) binding assay of TR-TUBE. Dynabeads conjugated to 2 μg of recombinant His-TR-TUBE were incubated with 2 μg of each diubiquitin for 30 min. All eight types of ubiquitin chain linkages, unbound and bound to TR-TUBE, were analyzed using SDS/PAGE and Coomassie Brilliant Blue (CBB) staining. The upper and lower arrows indicate TR-TUBE and diubiquitin, respectively. The asterisk denotes nonspecific protein eluted from Dynabeads. Sequence of FLAG-TR-TUBE (B) and its ubiquitin-binding–deficient mutant (C). The red and blue boxes indicate FLAG and mutated UBA domains within UBQLN1, respectively. Arg residues and adjacent Leu pairs, which were substituted with Ala residues, are indicated by the red circles and red boxes, respectively.

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WCL FLAG-tagged TR-TUBE mutant HA-tagged emp Skp2 emp Skp2 MG132 - ++ - ++ - ++ - ++ - - + - - + - - + - - + NEM

IP: αFLAG mutant TR-TUBE emp Skp2 emp Skp2 - ++ - ++ - ++ - ++ - - + - - + - - + - - +

kDa 188 98 62 49 38

Ubiquitin conjugates

28 17 14 6

αUbiquitin kDa

Ub

(Ub)n

250 150 100 75

-p27

50 37 25 20

p27

αp27 Fig. S2. Detection of ubiquitylated endogenous p27. 293T cells expressing FLAG-TR-TUBE or ubiquitin-binding–deficient FLAG-TR-TUBE mutant with HA-empty (emp) or HA-Skp2 were treated with or without MG132 and N-etylmaleimide (NEM; DUB inhibitor). Cell lysates obtained 48 h posttransfection were immunoprecipitated with anti-FLAG antibody, and WCLs and immunoprecipitates were analyzed by immunoblotting using anti-ubiquitin or anti-p27 antibody. IP, immunoprecipitation; Ub, ubiquitin.

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A

IP: αFLAG

B

IP: αFLAG

FLAG-TR-TUBE posttransfection (h) 24 48 72 emp Skp2 emp Skp2 emp Skp2 HA-tagged MG132 - + - + - + - + - + - +

C

IP: αFLAG FLAG-TR-TUBE 24 48 72 emp W7 emp W7 emp W7 - + - + - + - + - + - +

FLAG-TR-TUBE 24 48 72 emp Skp2 emp Skp2 emp Skp2 - + - + - + - + - + - + kDa 250

kDa 250

150 100 75 50

150 100 75

150 100 75

50

50

37

37

37

25 20

25 20

25 20

kDa 250

D

IP: αFLAG

E

IP: αFLAG

FLAG-TR-TUBE 24 48 72 posttransfection (h) emp W1 emp W1 emp W1 HA-tagged - + - + - + - + - + - + MG132 kDa 250 150 100 75 50 37 25 20

αNFKBIA

αc-Myc

αCDT1

αp27

F

IP: αFLAG FLAG-TR-TUBE 24 48 72 emp MDM2emp MDM2 emp MDM2 - + - + - + - + - + - +

FLAG-TR-TUBE 24 48 72 emp W1 emp W1 emp W1 - + - + - + - + - + - +

kDa 250

kDa 250

150 100 75 50

150 100 75 50

37

37

25 20

25 20

αPDCD4

αp53

Fig. S3. Time course of ubiquitination of endogenous substrates under expressing E3 and TR-TUBE. 293T cells were cotransfected with FLAG-TR-TUBE and HA-empty, WT F-box protein [Skp2 (A and B), FBXW7 (W7; C), and FBXW1 (W1; D and E)], or MDM2 (F), and the transfected cells were harvested at the indicated times. Cells were treated with or without MG132 for 4 h before harvesting. Anti-FLAG immunoprecipitates were analyzed by immunoblotting. Although ubiquitinated c-Myc was accumulated to high levels 24 h posttransfection in cells coexpressing FBXW7 (W7) and TR-TUBE, other ubiquitinated substrates were present at high levels 48 h posttransfection.

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Ub #PSM

HA-tagged

1000

(Ly sa te te d

) rea 2t

MG 13

1500

emp ΔF Skp2 emp ΔF Skp2 emp ΔF Skp2

TR -TU B

2000

TR -TU B

E( FL A

G

IP)

)

B E( Ly sa te

A

kDa 188

500

98 62 49 38

MG(diGly) MG132 (diGly)

28

p ΔF

emp DFSkp2

Sk p2

Ub-catcher TR-TUBE (diGly) (diGly)

em

emp DFSkp2

Sk p2

Ub-catcher TR-TUBE (Flag-diGly) (FLAG - diGly)

p ΔF

em

emp DFSkp2

em

Sk p2

p ΔF

0

17 14

Ub

6

αUbiquitin Fig. S4. Protection of polyubiquitin chain from trypsin by TR-TUBE. (A) Abundance of ubiquitin peptides identified in each experiment. The number of PSMs is used as a semiquantitative index. (B) Remaining amount of intact ubiquitin monomers or ubiquitin chains after trypsin digestion. The trypsinized lysates of TR-TUBE immunoprecipitates or WCLs from TR-TUBE–overexpressing or MG132-treated cells were analyzed by immunoblotting using anti-ubiquitin antibody.

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IP: αFLAG

A

IP: αFLAG MLN4924 Skp1

B

MLN4924

Skp1

- + - +

kDa

FLAG-F box

αTARS

75 37 100 75

FB X FB W1 X FB W7 X FB W8 X Sk W11 p FB 2 X em L12 p FB t y XO FB 5 X FB O7 X FB O9 X FB O11 X FB O21 X FB O22 XO 44

FLAG-FBXO21

kDa 75

αEID1

37

αFLAG

kDa 250

αTARS αEID1

150 100 75

αFLAG

50 37 25

SHP binding 1

54

EID1 1-187

120

pRB binding

IP: αHA

178 187

FLAG-FBXO21 binding +

1-177

+

1-168

-

1-157

-

52-187

+

62-187

+

116-187

not express

HA-EID1 kDa 37 25 20 100 75

1-187 1-177 1-168 1-157 52-187 62-187 116-187

C

αHA

αFLAG

Fig. S5. TARS and EID1 bind to FBXO21 in a specific manner. (A) 293T cells were transfected with 7 μg of FLAG-FBXO21 (-Skp1) expression plasmid alone or a combination of 3.5 μg of FLAG-FBXO21 plasmid and 3.5 μg of Skp1 plasmid. Thirty-four hours after transfection, cells were treated with or without 1 μM MLN4924 for 14 h. Anti-FLAG immunoprecipitates were analyzed by immunoblotting. The binding of FBXO21 with TARS and EID1 was stabilized by MLN4924 treatment and coexpression of Skp1. (B) 293T cells expressing FLAG-tagged F-box protein (shown in Fig. 2) and Skp1 were treated with MLN4924. Each antiFLAG immunoprecipitate was analyzed by immunoblotting. The FLAG-tagged F-box proteins tested immunoprecipitated as proteins of the predicted sizes. Both TARS and EID1 proteins were specifically detected in FBXO21 immunoprecipitates. (C) Schematic diagram of HA-tagged EID1 deletion mutants are shown at left. Cell lysates of 293T cells expressing FLAG-FBXO21 and each HA-tagged EID1 deletion mutant were immunoprecipitated with anti-HA, and immunoprecipitates were analyzed by immunoblotting. When cell lysates were immunoprecipitated with anti-FLAG antibody, coimmunoprecipitated HA-EID1 and its mutants could not be detected.

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(Ub)n

150 100 75

-TARS

αTARS kDa 250

(Ub)n -TARS

150 100 75

37

1-723 1-613 1-356 1-321 81-723 81-613 81-356 100-613 321-723

si FBXO21

emp F21 ΔF si F21 emp F21 ΔF si F21

FLAG-FBXO21

723

binding

HA-TARS

+ + + IP: αHA + + + not express +-

kDa 100 75 50 37

IP: αFLAG FLAG-TR-TUBE

G

αTARS

613

MLN4924

αTARS

1-723 1-613 1-356 1-321 81-723 81-613 81-356 100-613 321-723

321

cont FBXO21

kDa 100

N-extension N2 (Editing) Anticodon Binding N1 Aminoacylation 81 139

si cont

TARS mRNA 1.2

1.0 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 00

CHX (hr) 0 .5 1 2 3 0 .5 1 2 3 0 .5 1 2 3

25

αTARS

TARS

E

50 37

25 20

1

si

D Relative expression

kDa 250

C

em p FB ty FB XW1 XW FB 7 FBXW8 Sk XW1 p FB 2 1 emXL12 p FB ty X FB O5 X FB O7 X FB O9 FBXO1 X 1 FB O21 X FB O22 XO 44

FLAG-F box

50

F

IP: αHA HA-TR-TUBE

B

FLAG-TR-TUBE 6xMyc-tagged TARS emp F21 ΔF emp F21 ΔF HA-tagged - + - + - + - + - + - + MG132

co nt FB XO 21

IP: αFLAG

A

H2O2

-

- - - + + + +

kDa

αHA

75

αFLAG

75

αFLAG

250

(Ub)n

150

-TARS

100 75 50 37 25 20

IP: αFLAG 100

75 50 37

αHA

αTARS

H

H2O2

CHX (hr)

kDa 100

0 2 4 6 8 10

DMSO 0 2 4 6 8 10

αTARS

Fig. S6. FBXO21 recognizes and ubiquitinates the editing domain of TARS. (A) Ubiquitination assay of TARS. Cells expressing FLAG-TR-TUBE in combination with empty (emp), WT FBXO21 (F21), or dominant-negative mutant FBXO21 (ΔF) were treated with or without MG132. Anti-FLAG immunoprecipitates were analyzed by immunoblotting. Arrows show the positions of 6× Myc-TARS (Upper) and endogenous TARS (Lower). Some FBXO21 ubiquitination activity was detected toward endogenous TARS, and ubiquitination of exogenously expressed TARS by FBXO21 was efficiently detected after TR-TUBE IP, with or without proteasome inhibition by MG132. (B) Ubiquitination assay of TARS using TR-TUBE and various F-box proteins. 293T cells were transfected with plasmids encoding HA-TR-TUBE and each FLAG-tagged F-box protein. Anti-HA immunoprecipitates were analyzed by immunoblotting. Arrows show the position of TARS. (C) RNAi-mediated knockdown of FBXO21. Forty-eight hours after siRNA (si) transfection, TARS levels in WCLs were assessed by immunoblotting. cont, control. (D) Quantitative RT-PCR analysis. Total RNA was prepared from 293T cells 48 h after cells were transfected with control or FBXO21-specific siRNA. The data shown are representative of three independent experiments. (E) Forty-eight hours after siRNA transfection, cells were incubated with 2 μg/mL cycloheximide. In parallel, cells were treated with 1 μM MLN4924 for 1 h before addition of cycloheximide (CHX). Cells were harvested at the indicated times after cycloheximide treatment, and 30 μg of lysates was immunoblotted with anti-TARS antibody. TARS protein was stable in 293T cells, and neither knockdown of FBXO21 nor MLN4924 treatment affected its stability, as shown in C. (F) Schematic diagram of HA-tagged TARS deletion mutants is shown at left. Human TARS contains four structural domains similar to bacterial TARS, as well as a eukaryotic-specific N-extension domain (1). Cell lysates of 293T cells expressing FLAGFBXO21 and each HA-tagged TARS deletion mutant were immunoprecipitated with anti-HA or anti-FLAG antibody, and immunoprecipitates were analyzed by immunoblotting. The levels of immunoprecipitated mutants lacking the C-terminal half (1–356, 1–321, and 81–356) were very low, implying that the mutant proteins were unstable, because they cannot dimerize via the aminoacylation- and anticodon-binding domains. However, FBXO21 was clearly detected in the immunoprecipitates of C-terminal–deleted TARS mutant (1–356). By contrast, the 321–723 mutant, which contains the aminoacylation- and anticodon-binding domains, was stable but failed to bind to FBXO21. The deletion of the N-extension domain did not affect the interaction with FBXO21, but the N1 domain mutant (100–613) significantly reduced its binding ability. These interactions were confirmed by reciprocal immunoprecipitation with anti-HA antibody. (G) Thirty-six hours after siRNA and/or plasmid transfection, cells were treated with 2 mM H2O2 or DMSO for 30 min, washed twice, and cultured overnight. Cell lysates obtained 48 h posttransfection were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitates were analyzed by immunoblotting. Ubiquitination of TARS by SCFFBXO21 was slightly elevated in H2O2-treated cells. Knockdown of FBXO21 by siRNA treatment suppressed ubiquitination of TARS. (H) Cells were incubated with 2 mM H2O2 or DMSO for 30 min, washed twice with DMEM, and then incubated with cycloheximide. Cells were harvested at the indicated times after cycloheximide treatment, and lysates (30 μg) were immunoblotted with anti-TARS antibody. 1. Ruan ZR, et al. (2015) Identification of lethal mutations in yeast threonyl-tRNA synthetase which reveals critical residues in its human homolog. J Biol Chem 290(3):1664–1678.

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Table S1. Substrate ubiquitination sites identified in this study Ubiquitination site

Accession Substrates of Skp2 P46527

Annotation CDKN1B

Reported by MS

K153, K165 None

TARS

LNEKVNTPTTTVYR, K5(GlyGly) NSSTYWEGKADMETLQR, K9(GlyGly) FQEEAKNR, K6(GlyGly) HTGKIK, K4(GlyGly)

K243 K288 K319 K271

EID1

VSAALEEADKMFLR, K10(GlyGly); K133 M11(oxidation) SGAQQLEEEGPMEEEEAQPMAAPEGKR, M12(oxidation); M20(oxidation); K72 K26(GlyGly)

CDKN1A

Q9H211

CDT1

P61024

CKS1B

Q9Y6B2

This study

KRPATDDSSTQNKR, K1(GlyGly); K13(GlyGly) KRPATDDSSTQNKR, K13(GlyGly) QTSMTDFYHSKR, M4(oxidation); K11(GlyGly) LIFSKR, K5(GlyGly) IAPPKLACR, K5(GlyGly); C8(methylthio) SHKQIYYSDKYDDEEFEYR, amino-terminus(acetyl); K3(GlyGly) HVMLPKDIAK, M3(oxidation); K6(GlyGly)

P38936

Substrates of FBXO21 P26639

Identified peptide sequence

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K165 K154 K161 K24 K4

K75, K154

Reported by mutagenesis K134, K153, K165

K75, K141, K154, K161

K24, K141, K166, K189, K240, K307, None K356, K377, K416, K433, K522 K4, K11, K26 None

K26

K26, K65, K75, K91, K222, K243, K271, K279, K288, K306, K313, K319, K504, K529, K543, K549, K611,K636, K660, K681, K712, K717 K72, K133

None

None

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Table S2. F-box proteins identified in Cul1 immunoprecipitates from 293T cells Accession

F-box protein

Molecular mass, kDa

Known substrate

Other

Q9UK99 Q8NEZ5 Q9UK97 Q9Y3I1 Q9UKB1 Q13309 Q9NXK8 Q8TB52 O94952 Q8N3Y1 Q86XK2 Q9UKT4

FBXO3 FBXO22 FBXO9 FBXO7 FBXW11 SKP2 FBXL12 FBXO30 FBXO21 FBXW8 FBXO11 FBXO5

54.5 44.5 52.3 58.5 62.1 47.7 37.0 82.3 72.2 67.4 103.5 50.1

HIPK2, EP300, GTF2H1 KDM4A TTI1, TELO2 cIAP1 concerning Parkinson’s disease CTNNB1, IFNAR1, BST2 CDKN1B, CDKN1A, CDT1 etc Ku80, CaMKI Concerning BMP signaling

(1, 2) (3) (4) (5, 6) (7–9) (10) (11, 12) (13)

IRS-1, TBC1D3, GORASP1 BCL6, CDT2 Inhibitor for APC/C

(14–16) (17–19) (20, 21)

1. Shima Y, et al. (2008) PML activates transcription by protecting HIPK2 and p300 from SCFFbx3-mediated degradation. Mol Cell Biol 28(23):7126–7138. 2. Kainulainen M, et al. (2014) Virulence factor NSs of rift valley fever virus recruits the F-box protein FBXO3 to degrade subunit p62 of general transcription factor TFIIH. J Virol 88(6): 3464–3473. 3. Tan MK, Lim HJ, Harper JW (2011) SCF(FBXO22) regulates histone H3 lysine 9 and 36 methylation levels by targeting histone demethylase KDM4A for ubiquitin-mediated proteasomal degradation. Mol Cell Biol 31(18):3687–3699. 4. Fernández-Sáiz V, et al. (2013) SCFFbxo9 and CK2 direct the cellular response to growth factor withdrawal via Tel2/Tti1 degradation and promote survival in multiple myeloma. Nat Cell Biol 15(1):72–81. 5. Chang YF, Cheng CM, Chang LK, Jong YJ, Yuo CY (2006) The F-box protein Fbxo7 interacts with human inhibitor of apoptosis protein cIAP1 and promotes cIAP1 ubiquitination. Biochem Biophys Res Commun 342(4):1022–1026. 6. Di Fonzo A, et al. (2009) FBXO7 mutations cause autosomal recessive, early-onset parkinsonian-pyramidal syndrome. Neurology 72(3):240–245. 7. Suzuki H, et al. (1999) IkappaBalpha ubiquitination is catalyzed by an SCF-like complex containing Skp1, cullin-1, and two F-box/WD40-repeat proteins, betaTrCP1 and betaTrCP2. Biochem Biophys Res Commun 256(1):127–132. 8. Kumar KG, et al. (2003) SCF(HOS) ubiquitin ligase mediates the ligand-induced down-regulation of the interferon-alpha receptor. EMBO J 22(20):5480–5490. 9. Mangeat B, et al. (2009) HIV-1 Vpu neutralizes the antiviral factor Tetherin/BST-2 by binding it and directing its beta-TrCP2-dependent degradation. PLoS Pathog 5(9):e1000574. 10. Frescas D, Pagano M (2008) Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: Tipping the scales of cancer. Nat Rev Cancer 8(6):438–449. 11. Postow L, Funabiki H (2013) An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation. Cell Cycle 12(4):587–595. 12. Mallampalli RK, et al. (2013) Fbxl12 triggers G1 arrest by mediating degradation of calmodulin kinase I. Cell Signal 25(10):2047–2059. 13. Sartori R, et al. (2013) BMP signaling controls muscle mass. Nat Genet 45(11):1309–1318. 14. Xu X, et al. (2008) The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation. Mol Cell 30(4):403–414. 15. Kong C, et al. (2012) Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is mediated by CUL7 E3 ligase. PLoS ONE 7(9):e46485. 16. Litterman N, et al. (2011) An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning. PLoS Biol 9(5):e1001060. 17. Duan S, et al. (2012) FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas. Nature 481(7379):90–93. 18. Abbas T, et al. (2013) CRL1-FBXO11 promotes Cdt2 ubiquitylation and degradation and regulates Pr-Set7/Set8-mediated cellular migration. Mol Cell 49(6):1147–1158. 19. Rossi M, et al. (2013) Regulation of the CRL4(Cdt2) ubiquitin ligase and cell-cycle exit by the SCF(Fbxo11) ubiquitin ligase. Mol Cell 49(6):1159–1166. 20. Reimann JD, et al. (2001) Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex. Cell 105(5):645–655. 21. Wang W, Kirschner MW (2013) Emi1 preferentially inhibits ubiquitin chain elongation by the anaphase-promoting complex. Nat Cell Biol 15(7):797–806.

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Table S3. Candidates of FBXO21 substrates FLAG-TR-TUBE/ HA-empty Accession Experiment 1 P26639 P61158 Q9H040 Q6ZVT6 Q8IWX8 P24468 P43003 Q9Y6B2 Q15800 Q01650 P11908 Q9NR31 O94979 Q00059 Q9BZX2 Q9NNW5 Q7Z570 Experiment 2 P26639 P28330 Q9Y6B2 Q13867 Q9U.K.F6 Q9H305 P53985 O75380 P30405 O95997 P82909 Experiment 3 P26639 Q9Y6B2 Q13443 P14635 Q9NPA0 P31040 Q96EB1 Q9H000 P30405 P37802 Q3ZCQ8 Q96AY4

FLAG-TR-TUBE/ HA-FBXO21 ΔF

FLAG-TR-TUBE/HA-FBXO21

Gene name

Score

No. of PSMs

Score

No. of PSMs

Score

No. of PSMs

Molecular mass, kDa

TARS ACTR3 SPRTN C3orf67 CHERP NR2F2 SLC1A3 EID1 MSMO1 SLC7A5 PRPS2 SAR1A SEC31A TFAM UCK2 WDR6 ZNF804A

212.80

10

23.88

3

495.22 45.17 23.84 14.44 29.94 20.18 24.64 24.61 26.49 26.68 40.78 39.30 32.88 26.79 29.06 30.64 23.64

32 7 6 3 2 10 4 5 3 1 3 3 6 3 12 6 5

83.4 47.3 55.1 76.2 103.6 45.5 59.5 20.9 35.2 55.0 34.7 22.4 132.9 29.1 29.3 121.6 136.8

TARS ACADL EID1 BLMH CPSF3 CDIP1 SLC16A1 NDUFS6 PPIF PTTG1 MRPS36

335.59

14

120.24

9

555.12 32.48 601.21 41.02 34.06 26.55 23.02 22.22 33.08 21.99 21.51

30 2 25 4 2 5 7 1 1 1 1

83.4 47.6 20.9 52.5 77.4 21.9 53.9 13.7 22.0 22.0 11.5

TARS EID1 ADAM9 CCNB1 EMC7 SDHA ELP4 MKRN2 PPIF TAGLN2 TIMM50 TTC28

285.00

9

94.34

4

433.09 38.15 21.33 39.97 28.09 20.09 25.44 32.11 33.08 27.90 22.50 34.55

19 8 1 2 3 1 5 2 1 6 2 2

83.4 20.9 90.5 48.3 26.5 72.6 46.6 46.9 22.0 22.4 39.6 270.7

The extracted data of candidate proteins, whose scores and PSM numbers increased in cells expressing FBXO21 and decreased in cells expressing mutant FBXO21, are shown.

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