supporting information - pnas · 2015-03-31 · supporting information yoshida et al....

Supporting InformationYoshida et al. 10.1073/pnas.1422313112SI Materials and MethodsPlasmid Construction. To construct a polyubiquitin-binding probe,we modified a previously reported high-affinity probe for poly-ubiquitin, TUBE (1). cDNA encoding the ubiquitin-associated(UBA) domain of human UBQLN1 (NM_013438) was sub-cloned into a pBSKS (+) vector, and three Arg residues weresubstituted with Ala residues using a QuikChange Site-DirectedMutagenesis II Kit (Agilent). DNA encoding four tandem copiesof the UBA domains, joined by the flexible linker sequence(GlyGlyGlySerGlyGlyGly), was subcloned into vector pcDNA3-FLAG. 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 do-main of Dsk1 (2). To construct an ubiquitin-binding–deficientTR-TUBE mutant, we generated a synthetic gene encoding theTR-TUBE mutant in which two conserved Leu residues, corre-sponding to L368L369 of Dsk1, were replaced with Ala residuesin four positions simultaneously, and we then subcloned the re-sultant construct into pcDNA3-FLAG (Fig. S1C). cDNA en-coding Skp2 (NM_013787), FBXW7 (NM_033632), FBXW1/BTRC (NM_003939), FBXO21 (NM_015002), the ΔF mutantsof the preceding genes, MDM2 (XM_005268872), or CDKN1B(NM_004064) was subcloned into pcDNA3-HA. cDNA en-coding CKS1B (NM_001826) or TARS (NM_15295) was sub-cloned into pcDNA3.1-6× Myc. For FLAG-tagged F-box pro-tein 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 subclonedinto pcDNA3-FLAG.

Cell Culture and Transfection. 293T cells (1.3 × 106) were culturedin DMEM supplemented with 10% (vol/vol) FBS in a 10-cm cellculture dish for 24 h. Cells were transiently transfected with 3.5μg of FLAG-tagged expression plasmid (TR-TUBE or ubiquitin)and 3.5 μg of HA-tagged F-box protein plasmid with 21 μg ofpolyethylenimine (Polysciences).

Cell Viability Assay. 293T cells (1 × 105) were cultured in a six-wellcell culture plate for 24 h. Cells were transiently transfected with2 μg of plasmid. Harvested cells were suspended in cell culturemedium, and propidium iodide was added at a final concentra-tion of 2 μg/mL. After incubation for 5 min, the numbers ofstained or total cells were counted in “viability” mode on a TaliImage-Based Cytometer (Life Technologies).

In Vitro Diubiquitin-Binding Assay. cDNA encoding TR-TUBE wascloned into pRSET-A (Life Technologies), in which a Ser residueupstream of 6× His tag was substituted with a Cys residue topermit biotinylation. The resultant His-TR-TUBE was expressedin E. coli Rosetta2 (DE3) for 15 h at 22 °C. Bacterial cells werelysed by passage through a precooled French pressure cell (OhtakeWorks) 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. Thesupernatant was incubated with TALON resin (Clontech), andTR-TUBE was eluted with elution buffer [50 mM sodium-Hepes(pH 7.1), 100 mM NaCl, 0.2 M imidazole]. Then, TR-TUBEwas biotinylated with EZ-link Maleimide-PEG2-Biotin (ThermoScientific) and further purified by gel filtration on Superdex 75

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 His-

TR-TUBE was incubated with a 50-μL suspension of DynabeadsMyOne Streptavidin C1 (Life Technologies) for 30 min, and thebeads were washed twice. The conjugated TR-TUBE was in-cubated with 2 μg of each diubiquitin (M1, K6, K11, K27, K29,K33, K48, or K63; Boston Biochem) in 20 μL of 0.1% Triton-X100 in 50 mM ammonium bicarbonate for 30 min. After in-cubation, the supernatant (unbound fraction) was removed andthe beads were washed three times with the incubation buffer.The bound diubiquitin and TR-TUBE (bound fraction) wereeluted by boiling with SDS sample buffer. Both unbound andbound fractions were subjected to SDS/PAGE and stainedwith Coomassie Brilliant Blue.

Immunoprecipitation and Immunoblotting. To inhibit cellular pro-teasome activity, cells were treated with 10 μM MG132 (PeptideInstitute, Inc.) 4 h before harvesting. For TNF-α stimulation,cells were treated with 3 μg/mL TNF-α for 20 min before har-vesting. To inhibit NEDD8-activating enzyme activity, cells weretreated with 1 μM MLN4924 (Boston Biochem) 14 h beforeharvesting. Forty-eight hours after transfection, cells were lysedwith 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 werelysed with TBS-N in the presence of 1 mM N-ethylmaleimide.Lysates were centrifuged at 20,000 × g for 20 min at 4 °C. Thesupernatants [whole-cell lysate (WCL)] were immunoprecipitatedwith anti-FLAG monoclonal antibody (anti-DDDDK)-conjugatedagarose (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 immu-noblot analysis as previously described, with antibodies againstubiquitin (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, Rabbit44D4; Cell Signaling,), PDCD4 (1:1,000, Rabbit D29C6; Cell Sig-naling), 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 CruzBiotechnology), 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 UbiquitinatedProtein Identification. All steps were performed at 4 °C unlessotherwise noted. For FLAG-TR-TUBE immunoprecipitation,2 mL of WCL (∼1.5 mg/mL) prepared from a 10-cm cell culturedish harvested 48 h posttransfection (∼1 × 107 cells) was in-cubated for 1 h with 25 μL of anti-FLAG monoclonal antibody-conjugated agarose beads. After the agarose beads were washedfive times with 1.5 mL of TBS-N and twice with 1.5 mL of 50 mMammonium bicarbonate (AMBIC), bead-bound proteins wereeluted three times with 25 μL of 200 μg/mL FLAG peptide(Sigma) in 50 mM AMBIC. Proteins were reduced in 5 mMTCEP for 30 min at 50 °C and then alkylated with 10 mMmethylmethanethiosulfonate (MMTS) for 10 min at roomtemperature. Next, alkylated proteins were digested overnight at37 °C with 1 μg of trypsin (Promega). After tryptic digestion,samples were acidified to ∼pH 2 with TFA and desalted by

Yoshida et al. www.pnas.org/cgi/content/short/1422313112 1 of 11

www.pnas.org/cgi/content/short/1422313112

solid-phase extraction using GL-Tip SDB and GL-Tip GC (bothfrom GL Sciences).For direct immunoprecipitation of ubiquitinated peptides,

∼1 × 107 harvested cells were washed with PBS and then lysed bysonication 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 roomtemperature. Proteins in the supernatants were reduced in 5 mMTCEP for 2 h at 37 °C and alkylated with 10 mM MMTS for10 min at room temperature. The resultant solution was dilutedwith 3 vol of 20 mM Hepes (pH 8), and proteins were digestedovernight with 10 μg of trypsin at 37 °C with rotation. Aftertryptic digestion, samples were acidified to ∼pH 2 with TFA andcentrifuged at 20,000 × g for 15 min; the resultant supernatantwas applied to a Sep-PaK Light C18 cartridge (WAT023501;Waters) for peptide purification.

Immunoprecipitation of diGly-Containing Peptide. Ubiquitinatedpeptides were enriched by using the PTMScan Ubiquitin Rem-nant Motif (K-e-GG) Kit (Cell Signaling). Briefly, eluted pep-tides were dried by vacuum centrifugation, dissolved in 0.2 mL ofIAP buffer [50 mM Mops (pH 7.2), 10 mM Na2HPO4, 50 mMNaCl], adjusted to pH 7 with 1 M Tris, and incubated for 2 hat 4 °C with 10 μL of ubiquitin branch motif immunoaffinitybeads. Then, the beads were washed twice with 250 μL of IAPbuffer and three times with 250 μL of distilled water, andpeptides were eluted with 3 × 20 μL of 0.15% TFA. The elutedpeptides were desalted using GL-Tip SDB and GL-Tip GCbefore LC-MS analysis.

Immunoaffinity Purification and Trypsin Digestion for Cul1- or F-BoxProtein–Associated Protein Identification. Immunoprecipitationswere performed by incubating WCL (∼1.5 mg/mL) with anti-FLAG monoclonal antibody and Dynabeads Protein G (LifeTechnologies) for 1 h. After the Dynabeads were washed fourtimes with 200 μL of TBS-N, immune complexes were elutedthree times with 20 μL of FLAG peptide (200 μg/mL; Sigma) andboiled for 5 min in 50 mM AMBIC with 0.1% Rapigest (Milli-pore). Next, denatured proteins were alkylated with MMTS anddigested overnight at 37 °C with 1 μg of trypsin. After trypticdigestion, samples were incubated in 0.5% TFA at 37 °C for 1 hand then centrifuged at 20,000 × g for 15 min to remove Rapi-gest. The resultant supernatant was applied to GL-Tip SDB andGL-Tip GC.

MS Analysis. Desalted tryptic digests were analyzed by nanoflowultra-HPLC (Easy nLC; Thermo Scientific) coupled to a QExactive mass spectrometer (Thermo Scientific). The mobilephases were 0.1% formic acid in water (solvent A) and 0.1%formic acid in 100% acetonitrile (solvent B). Peptides were di-rectly loaded onto a C18 analytical column [ReproSil-Pur (3 μm,75-μm i.d., and 12-cm length); Nikkyo Technos] and separatedusing 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 flowrate of 300 nL·min−1. For ionization, 1.8 kV of liquid junctionvoltage and a capillary temperature of 250 °C were used. The QExactive mass spectrometer was operated in the data-dependentMS/MS mode, using Xcalibur software (Thermo Scientific), withsurvey scans acquired at a resolution of 70,000 at m/z 200. Thetop 10 most abundant isotope patterns with a charge ranging

from 2–4 were selected from the survey scans with an isolationwindow of 2.0 m/z and fragmented by higher-energy collisionaldissociation with normalized collision energies of 28. The max-imum ion injection times for the survey and MS/MS scans were60 ms, and the ion target values were set to 3e6 and 1e5 for thesurvey and MS/MS scans, respectively. Ions selected for MS/MSwere dynamically excluded for 5 s for diGly peptide identifica-tion 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 Knowl-edgebase (version 2012_10) using the Mascot search engine. Theprecursor and fragment mass tolerances were set to 10 ppmand 20 millimass units, respectively. Methionine oxidation, pro-tein amino-terminal acetylation, pyroglutamate formation, Ser/Thr/Tyr phosphorylation, and diglycine modification of Lys side chainswere set as variable modifications, and Cys methylthio modificationwas set as a static modification for database searching. Peptideidentification was filtered at a 1% false discovery rate. To identifyspecific substrates of ubiquitin ligases, the results of three individualsamples (cells expressing FLAG-TR-TUBE with empty vector, WTubiquitin ligase, or dominant-negative mutant ubiquitin ligase) wereassembled into one multiconsensus report using Proteome Discov-erer software. Cumulative protein scores were compared based onsumming the ion scores and the total numbers of identified se-quences (PSMs) of ubiquitinated peptides (peptides containing Lys-e-Gly-Gly).

RNAi Experiment. SMART pool: siGENOME FBXO21, Rb1, andMDM2 siRNAs and a nonspecific control duplex were purchasedfrom Dharmacon. Transfection of siRNAs into cells was ac-complished using Lipofectamine 2000 (Invitrogen) at a finalconcentration of 50 nM in six-well dishes.

Quantitative Real-Time PCR Assay.The cDNA was synthesized from1 μg of total RNA using a Transcriptor First Strand cDNASynthesis Kit (Roche). Quantitative PCR was performed usingLightCycler 480 Probes Master (Roche) in a LightCycler 480(Roche). Signals were normalized to the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). The forward andreverse primer pairs were 5′-GCAATGTACCCGGACCAG-3′and 5′-TGTCAAGCACCTTCAAAACAA-3′ for FBXO21,5′-AACGGAGCCTTGCTAACG-3′ and 5′-TCCTCGCTCTCGA-AGTCTG-3′ for EID1, 5′-ATATCAAATTGCCTGTGGAATT-AGT-3′ and 5′-TCCAAGGTACAATCTTCTTCCAG-3′ forTARS, and 5′-AGCCACATCGCTCAGACAC-3′ and 5′-GCC-CAATACGACCAAATCC-3′ for GAPDH.

Cycloheximide Chase Assay. 293T cells (1 × 105) in 6-cm cell cul-ture dishes were transfected with siRNA. The cells were thencultured for 48 h, followed by incubation with 2 μg/mL cyclo-heximide. For MLN4924 treatment, cells were treated with 1 μMMLN4924 for 1 h before cycloheximide addition. For oxidativestress, cells were incubated with 2 mM H2O2 for 30 min, washedtwice with medium, and then incubated with cycloheximide.After incubation for the indicated periods, the cells were washedwith PBS and suspended in TBS-N containing protease inhibitormixture. Lysates (30 μg) were subjected to immunoblot analyses.

1. Hjerpe R, et al. (2009) Efficient protection and isolation of ubiquitylated proteins usingtandem ubiquitin-binding entities. EMBO Rep 10(11):1250–1258.

2. Sasaki T, Funakoshi M, Endicott JA, Kobayashi H (2005) Budding yeast Dsk2 protein forms ahomodimer via its C-terminal UBA domain. Biochem Biophys Res Commun 336(2):530–535.



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. Dynabeadsconjugated 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, unboundand bound to TR-TUBE, were analyzed using SDS/PAGE and Coomassie Brilliant Blue (CBB) staining. The upper and lower arrows indicate TR-TUBE and diu-biquitin, respectively. The asterisk denotes nonspecific protein eluted from Dynabeads. Sequence of FLAG-TR-TUBE (B) and its ubiquitin-binding–deficientmutant (C). The red and blue boxes indicate FLAG and mutated UBA domains within UBQLN1, respectively. Arg residues and adjacent Leu pairs, which weresubstituted with Ala residues, are indicated by the red circles and red boxes, respectively.



αp27

IP: αFLAG

MG132NEM

HA-tagged

- - + - - +- + + - + +

Skp2

- - + - - +- + + - + +

Skp2

(Ub)n

-p27

- - + - - +- + + - + +emp Skp2

- - + - - +- + + - + +

Skp2TR-TUBE

WCL

emp emp emp

18898624938

2817

14

6

kDa

mutant mutant

25015010075

50

37

2520

p27

kDa

αUbiquitin

Ubiquitin

conjugates

Ub

TR-TUBEFLAG-tagged

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



- +MG132emp Skp2

- +HA-tagged

250

150100

75

50

37

2520

kDa

FLAG-TR-TUBE

IP: αFLAG

αp27

- +emp Skp2

- + - +emp Skp2

- +

posttransfection (h) 24 48 72

250

150100

75

50

37

2520

kDa

- +emp Skp2

- +

FLAG-TR-TUBE

IP: αFLAG

- +emp Skp2

- + - +emp Skp2

- +

24 48 72

αCDT1

250

150100

75

50

37

2520

kDa

- +MG132emp

- +HA-tagged

FLAG-TR-TUBE

IP: αFLAG

- +emp

- + - +emp

- +

posttransfection (h) 24 48 72W1

αPDCD4

250

150100

75

50

37

2520

kDa

- +emp

- +

FLAG-TR-TUBE

IP: αFLAG

- + - + - + - +

24 48 72MDM2emp MDM2 emp MDM2

αp53

250

150100

75

50

37

2520

kDa

- +emp W7

- +

FLAG-TR-TUBE

IP: αFLAG

- +emp

- + - +emp

- +

24 48 72W7 W7

W1 W1

αc-Myc

250

150100

75

50

37

2520

kDa

- +emp

- +

FLAG-TR-TUBE

IP: αFLAG

- +emp

- + - +emp

- +

24 48 72W1 W1 W1

αNFKBIA

A B

D

C

E F

Fig. S3. Time course of ubiquitination of endogenous substrates under expressing E3 and TR-TUBE. 293T cells were cotransfected with FLAG-TR-TUBE andHA-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 theindicated 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 ubiquitinatedsubstrates were present at high levels 48 h posttransfection.



0

500

1000

1500

2000

emp DF Skp2 emp DF Skp2 emp DF Skp2

Ub-catcher (Flag-diGly)

Ub-catcher(diGly)

MG(diGly)

Ub

#P

SM

Skp2ΔF

emp

MG132(diGly)

TR-TUBE (diGly)

TR-TUBE(FLAG - diGly)

Skp2ΔF

emp

Skp2ΔF

emp

A B

188

98

6249

38

28

17

14

6

kDa

Skp

2ΔFHA-tagged em

pTR

-TU

BE (F

LAG

IP)

Skp

2ΔFem

p

Skp

2ΔFem

p

TR-T

UBE

(Lys

ate)

MG

132

treat

ed (L

ysat

e)

αUbiquitin

Ub

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



75

37αEID1

αFLAG

αTARS

100

75

MLN4924 - + - +

- Skp1

FLAG-FBXO21

IP: αFLAGA B

250

150

10075

50

37

25

kDa

αEID1

αFLAG

αTARS

FBXW

1FB

XW7

FBXW

8FB

XW11

Skp2

FBXL

12em

pty

FBXO

5FB

XO7

FBXO

9FB

XO11

FBXO

21FB

XO22

FBXO

44

FLAG-F box

Skp1MLN4924IP: αFLAG

75

37

kDa

kDa

1 54 120

SHP binding

187178

pRB binding

EID1

1-187

1-177

1-168

1-157

52-187

62-187

116-187

binding

+

+

+

+

-

-

not express

IP: αHA

αHA

αFLAG

37

2520

100

751-

187

1-17

71-

168

1-15

752

-187

62-1

8711

6-18

7

HA-EID1

FLAG-FBXO21

kDa

C

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 acombination 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 μMMLN4924 for 14 h. Anti-FLAG immunoprecipitates were analyzed by immunoblotting. The binding of FBXO21 with TARS and EID1 was stabilized by MLN4924treatment and coexpression of Skp1. (B) 293T cells expressing FLAG-tagged F-box protein (shown in Fig. 2) and Skp1 were treated with MLN4924. Each anti-FLAG immunoprecipitate was analyzed by immunoblotting. The FLAG-tagged F-box proteins tested immunoprecipitated as proteins of the predicted sizes. BothTARS 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 immunoprecipitateswere analyzed by immunoblotting. When cell lysates were immunoprecipitated with anti-FLAG antibody, coimmunoprecipitated HA-EID1 and its mutants couldnot be detected.



A

- +emp F21

- + - +ΔF

- +emp F21

- + - +ΔF

- TARSFLAG-TR-TUBE

IP: αFLAG

250

150100

75

50

37

25

kDa

HA-tagged6xMyc-tagged

(Ub)n

-TARS

MG132

αTARS

20

si

αTARS

cont

FBXO

21

0

0.2

0.4

0.6

0.8

1

1.2

Rel

ativ

e ex

pres

sion

TARS mRNA

00.20.40.60.81.0

cont FBXO21

ECHX (hr) 0 .5 1 2 3

si cont si FBXO21 MLN4924

0 .5 1 2 3 0 .5 1 2 3

αTARS

DMSO

αTARS

H H O2 2

CHX (hr) 0 2 64 8 10 0 2 64 8 10

FBXW

1FB

XW7

FBXW

8FB

XW11

Skp2

FBXL

12em

pty

FBXO

5FB

XO7

FBXO

9FB

XO11

FBXO

21FB

XO22

FBXO

44

FLAG-F box

HA-TR-TUBEIP: αHA

αTARS

250

150100

75

50

37

25

kDa

empt

y

B C D

IP: αHAαHA

αFLAG

10075

5037

1-72

31-

613

1-35

6

81-7

2381

-613

81-3

5610

0-61

332

1-72

3

1-32

1

HA-TARS

FLAG-FBXO21

kDa

10075

5037

75

75

IP: αFLAG

αHA

αFLAG

1 81 139 321 613 723

TARS1-7231-6131-356

81-72381-61381-356

100-613321-723

1-321

N-extensionN1

N2 (Editing)Aminoacylation

Anticodon Binding

binding

+++

+

-

not express+

+-

-+

F

G

H2O2

emp

F21 ΔF

FLAG-TR-TUBE

IP: αFLAG

- +- +- +- +

si F

21em

pF

21 ΔF si F

21

αTARS

250

150

10075

50

37

25

kDa

20

(Ub)n

-TARS

(Ub)n

-TARS

100kDa

100kDa

Fig. S6. FBXO21 recognizes and ubiquitinates the editing domain of TARS. (A) Ubiquitination assay of TARS. Cells expressing FLAG-TR-TUBE in combinationwith empty (emp), WT FBXO21 (F21), or dominant-negative mutant FBXO21 (ΔF) were treated with or without MG132. Anti-FLAG immunoprecipitates wereanalyzed by immunoblotting. Arrows show the positions of 6× Myc-TARS (Upper) and endogenous TARS (Lower). Some FBXO21 ubiquitination activity wasdetected toward endogenous TARS, and ubiquitination of exogenously expressed TARS by FBXO21 was efficiently detected after TR-TUBE IP, with or withoutproteasome inhibition by MG132. (B) Ubiquitination assay of TARS using TR-TUBE and various F-box proteins. 293T cells were transfected with plasmids en-coding 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 datashown 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 cyclo-heximide treatment, and 30 μg of lysates was immunoblotted with anti-TARS antibody. TARS protein was stable in 293T cells, and neither knockdown ofFBXO21 nor MLN4924 treatment affected its stability, as shown in C. (F) Schematic diagram of HA-tagged TARS deletion mutants is shown at left. Human TARScontains four structural domains similar to bacterial TARS, as well as a eukaryotic-specific N-extension domain (1). Cell lysates of 293T cells expressing FLAG-FBXO21 and each HA-tagged TARS deletion mutant were immunoprecipitated with anti-HA or anti-FLAG antibody, and immunoprecipitates were analyzed byimmunoblotting. The levels of immunoprecipitated mutants lacking the C-terminal half (1–356, 1–321, and 81–356) were very low, implying that the mutantproteins were unstable, because they cannot dimerize via the aminoacylation- and anticodon-binding domains. However, FBXO21 was clearly detected in theimmunoprecipitates of C-terminal–deleted TARS mutant (1–356). By contrast, the 321–723 mutant, which contains the aminoacylation- and anticodon-bindingdomains, 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 domainmutant (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. Celllysates 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 theindicated 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.



Table S1. Substrate ubiquitination sites identified in this study

Ubiquitination site

Accession Annotation Identified peptide sequence This study Reported by MSReported bymutagenesis

Substrates of Skp2P46527 CDKN1B KRPATDDSSTQNKR, K1(GlyGly);

K13(GlyGly)K153, K165 None K134, K153, K165

KRPATDDSSTQNKR, K13(GlyGly) K165P38936 CDKN1A QTSMTDFYHSKR, M4(oxidation);

K11(GlyGly)K154 K75, K154 K75, K141,

K154, K161LIFSKR, K5(GlyGly) K161

Q9H211 CDT1 IAPPKLACR, K5(GlyGly);C8(methylthio)

K24 K24, K141, K166, K189, K240, K307,K356, K377, K416, K433, K522

None

P61024 CKS1B SHKQIYYSDKYDDEEFEYR,amino-terminus(acetyl); K3(GlyGly)

K4 K4, K11, K26 None

HVMLPKDIAK, M3(oxidation);K6(GlyGly)

K26

Substrates of FBXO21P26639 TARS LNEKVNTPTTTVYR, K5(GlyGly) K243 K26, K65, K75, K91, K222, None

NSSTYWEGKADMETLQR, K9(GlyGly) K288 K243, K271, K279, K288, K306,FQEEAKNR, K6(GlyGly) K319 K313, K319, K504, K529, K543,HTGKIK, K4(GlyGly) K271 K549, K611,K636, K660, K681,

K712, K717Q9Y6B2 EID1 VSAALEEADKMFLR, K10(GlyGly);

M11(oxidation)K133 K72, K133 None

SGAQQLEEEGPMEEEEAQPMAAPEGKR,M12(oxidation); M20(oxidation);

K26(GlyGly)K72



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

Accession F-box protein Molecular mass, kDa Known substrate Other

Q9UK99 FBXO3 54.5 HIPK2, EP300, GTF2H1 (1, 2)Q8NEZ5 FBXO22 44.5 KDM4A (3)Q9UK97 FBXO9 52.3 TTI1, TELO2 (4)Q9Y3I1 FBXO7 58.5 cIAP1 concerning Parkinson’s disease (5, 6)Q9UKB1 FBXW11 62.1 CTNNB1, IFNAR1, BST2 (7–9)Q13309 SKP2 47.7 CDKN1B, CDKN1A, CDT1 etc (10)Q9NXK8 FBXL12 37.0 Ku80, CaMKI (11, 12)Q8TB52 FBXO30 82.3 Concerning BMP signaling (13)O94952 FBXO21 72.2Q8N3Y1 FBXW8 67.4 IRS-1, TBC1D3, GORASP1 (14–16)Q86XK2 FBXO11 103.5 BCL6, CDT2 (17–19)Q9UKT4 FBXO5 50.1 Inhibitor for APC/C (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.



Table S3. Candidates of FBXO21 substrates

FLAG-TR-TUBE/HA-empty

FLAG-TR-TUBE/HA-FBXO21 ΔF FLAG-TR-TUBE/HA-FBXO21

Accession Gene name Score No. of PSMs Score No. of PSMs Score No. of PSMs Molecular mass, kDa

Experiment 1P26639 TARS 212.80 10 23.88 3 495.22 32 83.4P61158 ACTR3 45.17 7 47.3Q9H040 SPRTN 23.84 6 55.1Q6ZVT6 C3orf67 14.44 3 76.2Q8IWX8 CHERP 29.94 2 103.6P24468 NR2F2 20.18 10 45.5P43003 SLC1A3 24.64 4 59.5Q9Y6B2 EID1 24.61 5 20.9Q15800 MSMO1 26.49 3 35.2Q01650 SLC7A5 26.68 1 55.0P11908 PRPS2 40.78 3 34.7Q9NR31 SAR1A 39.30 3 22.4O94979 SEC31A 32.88 6 132.9Q00059 TFAM 26.79 3 29.1Q9BZX2 UCK2 29.06 12 29.3Q9NNW5 WDR6 30.64 6 121.6Q7Z570 ZNF804A 23.64 5 136.8

Experiment 2P26639 TARS 335.59 14 120.24 9 555.12 30 83.4P28330 ACADL 32.48 2 47.6Q9Y6B2 EID1 601.21 25 20.9Q13867 BLMH 41.02 4 52.5Q9U.K.F6 CPSF3 34.06 2 77.4Q9H305 CDIP1 26.55 5 21.9P53985 SLC16A1 23.02 7 53.9O75380 NDUFS6 22.22 1 13.7P30405 PPIF 33.08 1 22.0O95997 PTTG1 21.99 1 22.0P82909 MRPS36 21.51 1 11.5

Experiment 3P26639 TARS 285.00 9 94.34 4 433.09 19 83.4Q9Y6B2 EID1 38.15 8 20.9Q13443 ADAM9 21.33 1 90.5P14635 CCNB1 39.97 2 48.3Q9NPA0 EMC7 28.09 3 26.5P31040 SDHA 20.09 1 72.6Q96EB1 ELP4 25.44 5 46.6Q9H000 MKRN2 32.11 2 46.9P30405 PPIF 33.08 1 22.0P37802 TAGLN2 27.90 6 22.4Q3ZCQ8 TIMM50 22.50 2 39.6Q96AY4 TTC28 34.55 2 270.7

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



supporting information - pnas · 2015-03-31 · supporting information yoshida et al....

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