Received 5 April; accepted 10 July 2002; doi:10.1038/nature01045.

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Supplementary Information accompanies the paper on Nature’s website

(http://www.nature.com/nature).

AcknowledgementsWe thank L. Ritchie and J. Horner of DFCI mouse core for advice and assistance;S. Dymecki, H. Westphal, M. Oren, S. Lowe, D. Silver, C. Der & J. DeCaprio for advice andreagents; and D. Livingston, J. DeCaprio and W. Kaelin for comments on the manuscript.N.B. is supported by the ACS John Peter Hoffman Award and the Liss Family Fund grantfor research in pancreatic cancer. R.A.D. is an American Cancer Society Professor andrecipient of the Steven and Michele Kirsch Foundation Investigator Award. This work wassupported by grants from the NCI (National Cancer Institute) and ACS (American CancerSociety).

Competing interests statementThe authors declare that they have no competing financial interests.

Correspondence and requests for materials should be addressed to R.A.D.

(e-mail: ron_depinho@dfci.harvard.edu).

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SINAT5 promotes ubiquitin-relateddegradation of NAC1to attenuate auxin signalsQi Xie*, Hui-Shan Guo*, Geza Dallman*, Shengyun Fang†,Allan M. Weissman† & Nam-Hai Chua‡

* Laboratory of Molecular Cell Biology, Temasek Life Sciences Laboratory,National University of Singapore, 1 Research Link, 117604 Singapore† Regulation of Protein Function Laboratory, Center for Cancer Research,National Cancer Institute, Bethesda, Maryland 20892, USA‡ Laboratory of Plant Molecular Biology, Rockefeller University, 1230 YorkAvenue, New York, New York 10021, USA.............................................................................................................................................................................

The plant hormone indole-3 acetic acid (IAA or auxin) controlsmany aspects of plant development, including the production oflateral roots1–3. Ubiquitin-mediated proteolysis has a central rolein this process. The genes AXR1 and TIR1 aid the assembly of anactive SCF (Skp1/Cullin/F-box) complex that probably promotesdegradation of the AUX/IAA transcriptional repressors inresponse to auxin4–8. The transcription activator NAC1, a mem-ber of the NAM/CUC family of transcription factors, functionsdownstream of TIR1 to transduce the auxin signal for lateral rootdevelopment9. Here we show that SINAT5, an Arabidopsis homo-logue of the RING-finger Drosophila protein SINA, has ubiquitinprotein ligase activity and can ubiquitinate NAC1. This activity isabolished by mutations in the RING motif of SINAT5. Over-expressing SINAT5 produces fewer lateral roots, whereas over-expression of a dominant-negative Cys49 ! Ser mutant ofSINAT5 develops more lateral roots. These lateral root pheno-types correlate with the expression of NAC1 observed in vivo.Low expression of NAC1 in roots can be increased by treatmentwith a proteasome inhibitor, which indicates that SINAT5 targetsNAC1 for ubiquitin-mediated proteolysis to downregulate auxinsignals in plant cells.

To investigate NAC1 action, we carried out yeast two-hybridassays using NAC1 as a bait. One protein that interacted with NAC1showed extensive sequence homology to two C3HC4 RING-fingerproteins, SINA from Drosophila10 and SIAH from human11. Becausethe gene encoding this protein is located on chromosome 5 wedesignated this protein SINA of Arabidopsis thaliana 5 (SINAT5,AF480944; Fig. 1a). The identities between SINAT5 and SINA(M38384) and SIAH (U76247) are 33% and 36%, respectively.

When SINAT5 was used as the bait, it interacted with theArabidopsis AtUBC9A (AF480945; a protein that shows homologyto members of the yeast Ubc4/5 and human UbcH5 families),ubiquitin (data not shown) and with itself (Fig. 1b). The carboxy-terminal region was responsible for SINAT5 dimerization (data notshown), which is consistent with results reported for SIAH12,13. Weverified the dimerization of SINAT5 and interaction of the dimerwith NAC1 by in vitro pull-down assays (Fig. 1c, d).

We determined the expression profile of SINAT5 by generatingtransgenic plants carrying a fusion of the SINAT5 promoter and theb-glucuronidase gene (GUS). SINAT5–GUS was expressed in lowamounts in the vascular tissues of mature roots (Fig. 1e). But ontreatment with auxin, expression was also detected in lateral rootinitials and in the elongation zone of the main root (Fig. 1e). Thisroot expression pattern is very similar to that of NAC1 afterinduction by auxin (ref. 9 and Fig. 1e), which suggests thatSINAT5 and NAC1 function in the same types of cell. As forNAC1, expression of SINAT5 was induced by auxin but more slowly(Fig. 1f).

To investigate the subcellular localization of SINAT5, we con-structed a fusion of the green fluorescent protein (GFP) gene and

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SINAT5 placed under the control of a CaMV 35S promoter.Transient expression in onion epidermal cells showed that, whereasGFP was distributed in both the cytosol and the nucleus, GFP–SINAT5 was localized predominantly in the nucleus (Fig. 1g), as hasbeen reported for NAC1 (ref. 9).

As several proteins containing RING motifs function as E3ubiquitin ligases in vitro14–16, we determined whether SINAT5 alsohas E3 activity. We expressed SINAT5 in Escherichia coli as a fusionwith maltose-binding protein (MBP). In the presence of ubiquitin,E1 and E2, purified MBP–SINAT5 could carry out self-ubiquitina-tion, whereas control purified MBP was inactive (Fig. 2a). Asnegative controls, we constructed two mutant proteins, Cys49 !Ser (C49S) and His64 ! Tyr (H64Y), in which the RING domain

was disrupted (Fig. 1a). Neither mutant protein could undergo self-ubiquitination (Fig. 2b), suggesting that an intact RING motif isneeded for E3 activity. In addition, when present in a 5–10-foldexcess the C49S mutant protein inhibited the self-ubiquitinationactivity of wild-type SINAT5 (Fig. 2c). A 10-fold increase inamounts of E2 did not reduce this inhibition (data not shown),which indicated that the E2 was not being sequestered by the excessamount of the C49S mutant present. These results show that theSINAT5 E3 activity requires dimerization and that the inactive C49SSINAT5 mutant can act as a dominant-negative protein, probablyby forming inactive dimers with wild-type SINAT5.

The interaction between SINAT5, an E3 ligase, and NAC1suggests that the SINAT5 may ubiquitinate the NAC1 in vivo andtarget it for proteasomal degradation. To show directly that NAC1 isa substrate of SINAT5, we carried out ubiquitination experimentsusing Myc–NAC1, which was immunopurified from transgenicplants overexpressing the tagged protein. Myc–NAC1 was ubiqui-tinated by SINAT5 and this reaction was clearly dependent on E1, E2and SINAT5 acting as an E3 ligase (Fig. 2d).

In transgenic plants, the overexpression of NAC1 produces morelateral roots, whereas the underexpression of NAC1 produces fewerlateral roots9. If SINAT5 ubiquitinates NAC1 in vivo, therebypromoting its degradation, we would expect overexpression ofwild-type SINAT5 to reduce and overexpression of the dominant-negative C49S mutant to increase the numbers of lateral roots,respectively. These results were observed in transgenic experiments(Fig. 3). In addition, the main root length of SINAT5-overexpres-sing plants was longer than that of wild-type plants, whereas theopposite was true for C49S mutant plants. This effect of SINAT5 issimilar to that observed in the auxin-response mutant axr-1(ref. 1).In a separate experiment, 10-day-old seedlings (n ¼ 30) weretransferred from MS (Murashige and Shoog) medium to fresh MSmedium containing 2 mM 1-naphthaleneacetic acid (1-NAA) for

Figure 1 Molecular and cellular properties of SINAT5. a, Amino acid sequence

comparison of SINAT5, SINA and SIAH. Asterisks and triangles indicate conserved

cysteine and histidine residues, respectively, in the RING motif and the two zinc-fingers13.

b, SINAT5 interacts with NAC1, AtUBC9A and itself. ‘þ’ indicates cell growth and

b-galactosidase activity. c, Dimerization of SINAT5 in vitro. Recombinant MBP or

MBP–SINAT5 was mixed with in vitro translated SINAT5 (Methods). d, SINAT5 binds to

NAC1 in vitro. Recombinant MBP or MBP–SINAT5 was mixed with in vitro translated

NAC1. e, Expression pattern of SINAT5 and NAC1. Two-week-old transgenic seedlings

were treated with (þ) or without (2) 2 mM 1-NAA for 6 h before being processed for GUS

staining. Scale bar, 0.1 mm. f, Auxin induces expression of both NAC1 and SINAT5.

Cultured roots from wild-type seedlings9 were treated with 2 mM 1-NAA, and the RNA

expression at different times was analysed. g, SINAT5 is localized predominately in nuclei.

Cells were analysed by confocal microscopy after 16 h of incubation. Scale bar, 1 mM.

Figure 2 E3 activity of wild-type and mutant SINAT5. a, MBP–SINAT5 was assayed for E3

activity in the presence of E1, E2 and 32P-labelled ubiquitin (Ub) as indicated. b, MBP–

SINAT5 E3 activity is dependent on its RING motif. MBP–SINAT5 and mutants were

assayed for self-ubiquitination. c, SINAT5 (C49S) blocked the E3 activity of SINAT5.

Numbers indicate the relative amount of proteins present in the ubiquitination reaction,

where 1 equals 100 ng of MBP or MBP–SINAT. d, Ubiquitination of NAC1 by SINAT5.

Myc–NAC1 immunoprecipitated from transgenic plants was assayed for in vitro

ubiquitination. Samples were processed for western blot analysis using an antibody

against Myc.

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48 h. The mean ^ s.d. number of lateral roots per centimetre ofmain root was 13.4 ^ 0.4 for the vector control, 7.2 ^ 0.5 for plantsoverexpressing SINAT5, and 19.7 ^ 0.5 for plants overexpressingthe C49S mutant. These results show that SINAT5 inhibits auxin-induced lateral root development, whereas the C49S mutantpromotes this process.

The expression of NAC1 in roots is low but can be increasedconsiderably by the proteasome inhibitor MG 132 (Fig. 4a), whichsuggests that the low amount of protein detected is probably causedby continuous ubiquitin-mediated proteolysis. Pull-down assaysusing extracts prepared from transgenic plants showed that bothwild-type SINAT5 and the C49S mutant could interact with NAC1in plant cells (Fig. 4b). Consistent with the post-translationalregulation of NAC1 by SINAT5 in vivo, overexpression of wild-type SINAT5 resulted in a reduced amount of NAC1 in roots,whereas overexpression of the C49S mutant led to higher quantitiesof NAC1 (Fig. 4c). In these transgenic plants, the levels of NAC1transcript remained about the same. Treating the roots with auxincaused a reduction in the amount of NAC1 protein without anyapparent effect on the amounts of the 35S–Myc–NAC1 transcript(Fig. 4d), which indicates that the hormone triggers post-transla-tional proteolysis of the transcription activator.

Regulated proteolysis is important in the auxin signalling path-way6,7. In the absence of auxin, the target genes for auxin signaltransduction are presumably repressed by AUX/IAA transcriptionalregulators. But on exposure to the hormone, these repressors aredegraded by ubiquitin-dependent proteolysis mediated by the SCFcomplex, of which TIR1 is an integral subunit, leading to anupregulation of auxin signals. We have shown here that anotherstep of the auxin signalling pathway is regulated by ubiquitin-mediated proteolysis. In this latter step, however, the ubiquitinationis mediated not by an SCF complex but by the RING motif proteinSINAT5, which shares extensive sequence homology with the SINA/

SIAH family of RING proteins10,11. Much evidence supports theview that SINAT5 targets NAC1 for post-translational degradation:first, the amount of NAC1 protein in vivo can be increased by theproteosome inhibitor MG 132; second, NAC1 can be ubiquitinatedby SINAT5 in vitro; third, NAC1 and SINAT5 are expressed in thesame types of cell and interact in vivo; and last, overexpression ofwild-type SINAT5 in transgenic plants leads to a decrease in theamount of NAC1 in roots and a concomitant reduction of lateralroot numbers, whereas the opposite is found in transgenic plantsoverexpressing the dominant-negative C49S mutant of SINAT5.The opposing effects of SINAT5 and the C49S mutant are alsoobserved when lateral root development is induced by auxin,thereby implicating the E3 ligase in this hormonal response.

Expression of both the SINAT5 and NAC1 genes is induced byauxin, although the former induction is slower. This difference inthe induction kinetics could explain the auxin-induced degradationof NAC1, which becomes apparent at 8 h after hormone treatment(Figs 1f and 4d). The Drosophila protein SINA promotes degra-dation of the cell-fate repressor Tramtrack17, whereas mammalianSIAHs control the stability of the membrane receptor Deleted inColorectal Cancer18, several transcription factors19,20 and otherproteins21–24. Our findings here provide insight into the functionof the SINA/SIAH family as E3 ligases. The capacity of a SINAT5protein containing a mutation in the RING motif to function as a

Figure 3 Phenotypes of transgenic plants. Seedlings of wild-type Landsberg ecotype

(Control), transgenic lines carrying a 35S–SINAT5 transgene, SINAT5 (WT), and

transgenic plants carrying a 35S–SINAT5(C49S) transgene, SINAT5 (C49S), were grown

vertically on MS agar medium containing 2% sucrose. Representative plants were

photographed after 10 d. Scale bar, 0.5 cm. The mean (^s.d.) number of lateral roots per

seedling and the mean length of main root per seedling (in parentheses) were determined

for 30 seedlings of each type: control, 6.2 ^ 0.3 (3.4 ^ 0.4 cm); SINAT5 (WT),

2.7 ^ 0.4 (4.6 ^ 0.4 cm) and SINAT5 (C49S), 10.9 ^ 0.2 (2.5 ^ 0.3 cm).

Figure 4 Relationship between amounts of SINAT5 and NAC1 proteins in transgenic

plants. a, Expression of NAC1 is increased by treatment with MG 132. Root cultures from

a Myc–NAC1 transgenic line were treated with 50 mM MG 132 (þ) or DMSO (2) for 3 h.

Protein extracts were processed for western blot analysis. b, SINAT5 interacts with NAC1

in vivo. Myc-tagged proteins from the roots of double transgenic plants were

immunoprecipitated and processed for western blot analysis. c, Relative amounts of

SINAT5 and NAC1 in transgenic roots. Proteins from the roots of transgenic plants were

processed for western blot analysis. Total RNAs from the same transgenic lines were

analysed by northern blot. d, Auxin promotes the degradation of Myc–NAC1. Root cultures

from a Myc–NAC1 transgenic line were treated with 2 mM 1-NAA.

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dominant-negative protein during in vitro ubiquitination suggeststhat the RING-independent dimerization of this protein family isfunctionally important for the E3 activity. Dimerization of RING-finger proteins as a means of activating E3 activity is in agreementwith results obtained with heterodimers of BRAC1 and BARD1(ref. 25).

In contrast to TIR1, which activates auxin signalling by proteindegradation6, SINAT5 attenuates the signal by targeting NAC1 fordegradation, thereby resetting the transduction cascade. The tran-scriptional activation of both NAC1 (ref. 9) and SINAT5 (Fig. 1e) byauxin indicates the importance of antagonistic transcriptional aswell as post-transcriptional mechanisms in fine-tuning NAC1-regulated aspects of auxin signalling. At present we do not knowwhether other modifications of NAC1 precede its ubiquitinationand how the self-ubiquitinating activity of SINAT5 and its ability toubiquitinate NAC1 are regulated by auxin. The functions of fourother SINAT family members in the Arabidopsis genome are also notknown. Future investigations into these and other related molecularevents will increase our understanding of the expanding role ofubiquitin-mediated proteolysis in cell signalling. A

MethodsYeast two-hybrid screensWe synthesized complementary DNAs from 5 mg of poly(A) RNA isolated from plants atdifferent growth stages using a commercial kit (Stratagene). The resulting double-stranded DNAs, with EcoRI and XhoI cut ends (average size 1.2 kilobases), were ligated to apGAD-GH vector (Clontech) that had been digested with EcoRI and XhoI for 48 h at 8 8C.After ligation, the cDNA library was used to transform E. coli strain DH10B (Gibco) byelectroporation. The cDNA library contains about 6 £ 106 primary transformants. Wealso isolated plasmids by standard methods. The yeast strain HF7c (MATa ura3-52his3-200 ade2-101 lys2-801 trp1-901 leu2-3, 112 gal4-542 gal80-538 LYS2::GAL1UAS–GAL1TATA–HIS3 URA3::GAL4 17mers( £ 3)–CyC1TATA–LacZ), containing the tworeporter genes lacZ and HIS3, were used. Yeast cells were transformed sequentially withpGB-NAC1(1–199), a plasmid containing sequences encoding the first 199 amino acids ofNAC1 fused to the Gal4 DNA-binding domain (a TRP1 marker) in the pGBT8 vector, andthen with the Arabidopsis cDNA library constructed as described above. We carried out thescreening as described26.

Transgenic plantsA binary vector VIP96 (KanR in plants) carrying a 35S–6 £ Myc–NAC1 transgene was usedto transform A. thaliana Landsberg ecotype. Wild-type plants and homozygous plantsoverexpressing Myc–NAC1 were retransformed with pBA002 (Basta resistance in plants)27

carrying either 35S–SINAT5 or 35S–SINAT5(C49S). We generated all mutations using theQuick-Change site-directed mutagenesis kit (Stratagene). For the SINAT5 promoter andGUS construct, we amplified by polymerase chain reaction (PCR) a 2-kilobase fragmentupstream from the ATG codon of SINAT5 and cloned it into pBI101 (Clontech).Arabidopsis plants were transformed by the floral dip method28 and transformants wereselected on MS plates containing 50 mg ml21 kanamycin and/or 10 mg ml21 Basta. Theconditions for plant growth, induction of auxin and GUS staining have been described9.For the cellular localization of SINAT5, plasmid DNA containing a 35S–SINAT5–GFPconstruct was bombarded into onion peels as described9.

Protein preparation, in vitro binding and ubiquitination assaysWe cloned DNA encoding SINAT5 and the SINAT5 (C49S) mutant into pMal-c2 (NewEngland Biolabs) and prepared the fusion proteins according to the manufacturer’sinstructions. [35S]methionine-labelled SINAT5 and NAC1 were generated by in vitrotranscription and translation with wheat-germ extracts using a T7/T3 coupled TnT kit(Promega). For in vitro binding, 2 ml of the translation mix was added to 200 ml of bindingbuffer containing 50 mM HEPES (pH 7.5), 1 mM EDTA, 150 mM NaCl, 10% glycerol,0.1% Tween 20 and 0.5 mM dithiothreitol (DTT), and the mixture was incubated at roomtemperature 25 8C for 1 h. After incubation, the beads containing amylose resin werewashed three times with washing buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl and0.2% Nonidet P-40). 32P-labelled ubiquitin was prepared using recombinant glutathioneS-transferase (GST)-labelled ubiquitin as described14,16. Unlabelled ubiquition (10 mg perreaction) was purchased from Sigma. Preparation of wheat E1 (the cDNA clone was a kindgift from R. Vierstra) and UbcH5B (E2) has been described14,16. For in vitro ubiquitination,MBP-labelled SINAT5 and/or the C49S mutant (500 ng) were immobilized on amyloseresin beads. Ubiquitination assays were carried out by adding 20 ng each of recombinantwheat E1 and UbcH5B, and 2 £ 104 c.p.m. of 32P-labelled ubiquitin in ubiquitinationbuffer containing 50 mM Tris (pH 7.4), 2 mM ATP, 5 mM MgCl2, 2 mM DTT. The reactionmix (30 ml) was incubated for 1.5 h at 30 8C with agitation in an Eppendorf Thermomixer.After the reaction, samples were heated to 95 8C in an SDS–PAGE sample buffer containingb-mercaptoethanol before being separated by electrophoresis on 10% SDS gels.

Western and northern blot analysesWe raised antibodies to SINAT5 in rabbits using as an antigen a synthetic 14-residue

peptide (TDSIDSVIDDDEIH) corresponding to amino acids 3–18 of SINAT5. Antibodiesto c-Myc (SC40 and SC40-AC) and actin (SC1616) from Santa Cruz Biotechnology wereused for immunoprecipitation and/or western blotting. Each lane contained 10 or 20 mgprotein. We extracted RNA using an RNeasyR Plant Mini kit (Qiagene). Analysis bynorthern blot has been described9, using as probes an NAC1 cDNA fragment encoding theNAC1 C-terminal region (amino acids 200–324) and a SINAT5-specific EcoRI/NdeIfragment. The 18S rRNA was used as a loading control. Each lane contained 10 mgRNA.

Received 13 February; accepted 25 June 2002; doi:10.1038/nature00998.

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AcknowledgementsWe thank E. Ng and W.-P. Tang for technical assistance; Y.-S. Chan for taking the GUSpictures; and P. Hare for reading the manuscript. This work was supported in part by agrant from the NIH to N.-H.C. This work was initiated in the Institute of MolecularAgrobiology, Singapore, supported by Singapore A-star funding.

Competing interests statementThe authors declare that they have no competing financial interests.

Correspondence and requests for materials should be addressed to N.-H.C.

(e-mail: chua@mail.rockefeller.edu).

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NATURE | VOL 419 | 12 SEPTEMBER 2002 | www.nature.com/nature170 © 2002 Nature Publishing Group