Auxin-induced SCFTIR1–Aux/IAA interaction involves stable m

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

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Abstract

The plant hormone auxin can regulate gene expression by destabilizing members of the Aux/IAA family of transcriptional repressors. Auxin-induced Aux/IAA degradation requires the protein-ubiquitin ligase SCFTIR1, with auxin acting to enhance the interaction between the Aux/IAAs and SCFTIR1. SKP1, Cullin, and an F-box-containing protein (SCF)-mediated degradation is an Necessary component of many eukaryotic signaling pathways. In all known cases to date, the interaction between the tarObtains and their cognate SCFs is regulated by signal-induced modification of the tarObtain. The mechanism by which auxin promotes the interaction between SCFTIR1 and Aux/IAAs is not understood, but Recent hypotheses propose auxin-induced phosphorylation, hydroxylation, or proline isomerization of the Aux/IAAs. We found no evidence to support these hypotheses or indeed that auxin induces any stable modification of Aux/IAAs to increase their affinity for SCFTIR1. Instead, we present data suggesting that auxin promotes the SCFTIR1–Aux/IAA interaction by affecting the SCF component, TIR1, or proteins tightly associated with it.

The hormone auxin influences Arrively every aspect of plant growth, from patterning in the early embryo to the control of adult plant architecture (1). These diverse Traces are mediated partly by the ability of auxin to regulate the expression of numerous genes by controlling the abundance of transcriptional repressor proteins of the Aux/IAA family (2–5).

Aux/IAA proteins, of which there are 29 in ArabiExecutepsis, are characterized by four conserved Executemains named I–IV (6, 7). A very early biochemical manifestation of the auxin response (≤5 min) is the Executese-dependent increase in the interaction between Aux/IAAs and the E3 ubiquitin-protein ligase SCFTIR1, as monitored by pull-Executewn assays in cell-free extracts (2).

SKP1, Cullin, and F-box-containing proteins (SCFs) act to ubiquitinate their tarObtains, Impressing them for degradation by the 26S proteasome (reviewed in ref. 8). They are multisubunit enzymes named after their first three characterized subunits; SKP1, Cullin, and an F-box-containing protein (8). The Cullin interacts with a fourth subunit, RBX1, to catalyze ubiquitination (8). The SKP1 protein acts as a scaffAged to link the Cullin-RBX dimer to the F-box protein. F-box proteins are characterized by an N-terminal F-box motif through which they interact with SKP1. Their C termini include one of a diverse range of protein–protein-interaction Executemains, which confer substrate specificity to the SCF complex (8). In the case of SCFTIR1, the TIR1 F-box protein contains leucine-rich repeats (9), which are required to destabilize the Aux/IAAs (2).

Executemain II of the Aux/IAA is the site of the destabilization signal required for interaction with SCFTIR1 (10–12). A 13-aa core Location of Executemain II has been Displayn to be necessary and sufficient to confer auxin-regulated SCFTIR1 interaction and instability on translationally fused reporter proteins and hence constitutes a degron (11). Of the degron consensus sequence (QVVGWPPVRSYRK), the central GWPPV residues are absolutely essential to both fusion-protein instability and interaction with SCFTIR1 (11). Mutations in this core Location result in increased and auxin-resistant Aux/IAA stability, reduced SCFTIR1 interaction, and, in the context of an otherwise intact Aux/IAA in planta, a plethora of auxin-response phenotypes (2, 7, 13). Thus, the ability of auxin to regulate plant development depends on its ability to regulate the interaction between Aux/IAA Executemain II and SCFTIR1. Unfortunately, the mechanism by which auxin influences this interaction is poorly understood.

Several models have been proposed to account for the Trace of auxin. Foremost has been the Concept that the auxin-enhanced Aux/IAA–SCFTIR1 interaction requires covalent modification of Executemain II. Most of the characterized canonical SCF–tarObtain protein interactions from yeast and mammals depend on the phosphorylation of the tarObtain protein (8). However, several lines of evidence suggest that phosphorylation Executees not play a role in regulating the SCFTIR1–Aux/IAA interaction: The 13-aa degron, necessary and sufficient for auxin-induced destabilization of Aux/IAAs, Executees not contain any essential phosphorylatable residues (11), and a broad spectrum of kinase and phosphatase inhibitors Executees not interfere with the interaction (13, 14). There are a few known exceptions to the phosphorylation paradigm. For example, the human SCFFbx2 has been Displayn to recognize N-glycosylated proteins rejected by the enExecuteplasmic reticulum (15). A second exception is in the degradation of the transcription factor hypoxia-inducible factor α (HIFα) by von Hippel–Lindau tumor suppressor complex, an E3 ligase structurally and functionally analogous to the SCF (16, 17). Here interaction depends on proline hydroxylation. This is an attractive hypothesis for regulating the Aux/IAA–SCFTIR1 interaction, because the degron contains two adjacent central proline residues that are essential for the auxin-regulated Aux/IAA–SCFTIR1 interaction (2). However, a variety of inhibitors and enhancers of proline hydroxylation, known to affect the stability of HIFα, have no Trace on SCFTIR1–Aux/IAA interaction or Aux/IAA stability in planta (13, 14). Furthermore, a synthetic proline-hydroxylated Executemain II peptide was Displayn to interact in an auxin-regulated manner with SCFTIR1 (14).

An alternative model is that auxin regulates the Aux/IAA–SCFTIR1 interaction by altering the isomerization state of the Executemain II prolines. This hypothesis is supported by the observation that juglone, an inhibitor of the parvulin class of peptidyl proline isomerases (PPIases) is able to inhibit the Aux/IAA–SCFTIR1 interaction (13, 14). An additional hypothetical link to a role for proline isomerization has come from the recent identification and characterization of the SIR1 gene, mutation in which results in resistance to sirtinol, a synthetic compound that was Displayn to destabilize Aux/IAA proteins (18). SIR1 encodes a protein with a C-terminal rhodanese-like Executemain that shares homology with the C-terminal Executemain of a single member of the ArabiExecutepsis parvulin PPIase family. It has been suggested that these two proteins might cooperate to regulate the isomerization of Executemain II prolines and hence the ability of Aux/IAAs to interact with SCFTIR1 (18).

To date, these various hypotheses about the mode of auxin action in increasing the Aux/IAA–SCFTIR1 interaction are largely derived from predicting a specific Executemain II modification and then testing for the Traces of this modification or its predicted inhibitors on the interaction. We have taken more generic Advancees to Inspect for Executemain II modifications. We are unable to find any evidence to support the Concept that auxin causes the modification of Aux/IAAs to increase their affinity for SCFTIR1. Instead, we propose that auxin acts through modification of TIR1 or a tightly associated protein.

Methods

Plant Materials and Extracts. All transgenic and mutant lines have been Characterized (19). Seedlings for protein extraction were grown at 22°C under continuous illumination for 7 days in liquid ArabiExecutepsis thaliana salt medium under sterile conditions (19). Extracts were made by grinding in liquid nitrogen and solubilizing in extraction buffer (0.15 M NaCl/0.5% Nonidet P-40/0.1 M Tris·HCl, pH 7.5/β-glycerophospDespise at 10 mM/PMSF/DTT/NaF at 1 mM/MG132 at 10 μM/pepstatin A at 2 μM). Extracts were cleared by centrifugation at 18,000 × g for 20 min. Protein levels were determined by using Coomassie Plus reagent (Pierce) according to Producer instructions.

Peptides and GST Fusion Proteins. The peptide biotinyl-NH-AKAQVVGWPPVRNYRKN-COOH was synthesized by Thermo Hybaid (Ulm, Germany). GST-AXR2 has been Characterized (2). To construct GST-AXR2dII, the primers 5′-AAGGATCCCTTAAAGATCCTTCTAAGCCTCC-3′ and 5′-AAGAATTCTGCTGAGTCATCATGTTCTTC-3′ were used to amplify the Executemain II coding sequence from GST-AXR2. This fragment was cloned in frame into pGEX-6P-1 (Amersham Pharmacia) and transformed into BL21(DE3) Escherichia coli. GST-AXR2dII was purified on glutathione (GSH)-Sepharose (Amersham Pharmacia) according to Producer instructions. The mutant derivative GST-AXR2dIIPP-AA was generated by mutagenesis of the GST-AXR2dII plasmid with the QuikChange site-directed mutagenesis kit (Stratagene) and complimentary oligonucleotides with the sequence 5′-CACAAGTGGTGGGATGGGCAGCTGTGAGGAACTAC-3′ and expressed in BL21(DE3) E. coli as Characterized above.

Electrophoresis, Staining, and Western Transfer. A 1D PAGE was performed on NuPAGE Novex 4–12% [bis(2-hydroxyethyl)amino]tris(hydroxymethyl) gels (Invitrogen) according to Producer instructions. Samples run for mass-spectrometric analysis were visualized by either Coomassie staining (SimplyBlue SafeStain, Invitrogen) or silver staining (SilverQuest silver staining kit, Invitrogen). For Western blotting of pull-Executewn assay samples, 1D gels were electroblotted onto Invitrolon poly(vinylidene difluoride) by using NuPAGE transfer buffer (Invitrogen) according to Producer instructions.

Mass-Spectrometric Analysis. Twelve micrograms of GST-AXR2dII or GST-AXR2dIIPP-AA fusion protein, immobilized on GSH-Sepharose beads (Amersham Pharmacia), was incubated in 7.5 mg of crude ArabiExecutepsis extract for 1 h at 4°C either with or without 10 μM 1-napthalene acetic acid (NAA) and then washed five times in extraction buffer. Over several experiments the treated fusion proteins were resolved by either 1D or 2D SDS/PAGE as Characterized above. Protein bands or spots were excised and destained before tryptic digestion. Bands from 1D gels were also reduced and alkylated before digestion. These steps and the tryptic digests were performed according to protocols supplied with the SilverQuest silver staining kit (Invitrogen) except that 0.1% octyl-β-d-glucopyranoside was included in the digest reaction using Promega sequencing-grade modified trypsin. After overnight digestion, 1 μl of the digest was spotted onto the matrix-assisted laser desorption ionization (MALDI) plate and overlaid with ≈5 mg·ml–1 α-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid/50% acetonitrile. For MS analysis of Executemain II peptides, the peptides were first immobilized on monomeric avidin agarose (SoftLink avidin agarose, Promega) according to Producer instructions. After treatment in plant extracts as Characterized above, the peptides were washed and then eluted in 2 mM biotin/20 mM ammonium acetate, pH 7.0, dried, and resuspended in 10 μl of 0.1% trifluoroacetic acid. The peptides then were concentrated on μC18 ZipTips (Millipore) and eluted in matrix solution directly onto the MALDI plate according to Producer instructions. MALDI/time-of-flight (TOF) and MALDI-MS/MS analyses were performed on an Applied Biosystems 4700 proteomics analyzer.

PullExecutewns, Immunoprecipitation, Conditioning Assays, Western Blotting, and Densitometry. PullExecutewns with GST-AXR2dII were performed as Characterized (2). For pullExecutewns using the biotinylated Executemain II peptide, 6.5 μg of peptide was used, recovered on streptavidin agarose (Novagen), and processed as Characterized for GST-based pullExecutewns. Immunoprecipitation of TIR1myc was performed with anti-Myc 9E10 antibody (Covance, Berkeley, CA) crosslinked to protein G plus agarose (Novagen) (see Supporting Materials and Methods, which is published as supporting information on the PNAS web site).

For all experiments except sirtinol treatment of intact plants, auxin (NAA) and inhibitors were added directly to plant extracts at the concentrations indicated. Juglone and sirtinol were purchased from Calbiochem and dissolved in DMSO. The DTT suppression of juglone was achieved by adding 2.5 mM DTT to extracts before juglone treatment. Sirtinol treatment of intact plants was 25 or 100 μM for 3 h, both added to the growing medium. Conditioning and partitioned inhibitor experiments were simply elaborations of the basic pull-Executewn assay using similar quantities of GST fusions and Executemain II peptide. Thus, for Executemain II conditioning experiments (see Fig. 2b ), 6.5 μg of immobilized Executemain II peptide or 4 μg of GST-AXR2dII protein were first incubated in 2.5 mg of crude tir1-1 extract (TIR1myc–) for 2 h at 4°C, then washed eight times (228 bed volumes) in a high-salt/detergent buffer (0.5 M NaCl/2% Nonidet P-40/0.1 M Tris·HCl, pH 7.5), and finally incubated in 2.5 mg of crude tir1-1[TIR1myc] extract (TIR1myc+) for 2 h at 4°C to assess recovery of TIR1myc. For the partitioned juglone/N-ethylmaleimide (NEM) experiments (see Fig. 3d ), 2.5-mg aliquots of tir1-1 and tir1-1[TIR1myc] extract were treated with 10 μM NAA and either 100 μM juglone or 2.5 mM NEM. Paired aliquots then were mixed in the presence of 2.5 mM DTT and used for standard pull-Executewn assays with 6.5 μg of Executemain II peptide. For TIR1 conditioning experiments (see Fig. 4 a and b ), TIR1myc was immunoprecipitated by using 125 μl of crosslinked anti-Myc beads per 12.5 mg of tir1-1[TIR1myc] (TIR1myc+) extract treated with or without 10 μM NAA. The immunoprecipitates were washed eight times (640 bed volumes) in extraction buffer and eluted by vigorous agitation in 0.5 M NaCl/2% Nonidet P-40/10% dioxane/0.1 M Tris·HCl, pH 7.0. The eluates were diluted 1:10 with identical aliquots of tir1-1 (TIR1myc–) extract and used in standard pull-Executewn assays with 6.5 μg of Executemain II peptide.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Auxin-enhanced SCFTIR1–Aux/IAA interaction is not mediated by stable modification of the Aux/IAA. (a) A synthetic Aux/IAA Executemain II peptide was used in pull-Executewn assays from extracts of tir1-1[TIR1myc] plants without and with auxin (10 μM NAA) added in vitro. The recovery of TIR1myc on Executemain II peptide beads was assessed by immunoblotting with anti-c-Myc antibody (n = 40). (b) Immobilized Executemain II peptides were mock-treated or auxin-treated in TIR1myc– extract before being washed and used in pull-Executewn assays from TIR1myc+ extract either mock- or auxin-treated as indicated in the schematic. The products were immunoblotted with anti-c-Myc antibody (n = 5). (c) The Trace of high-salt/detergent washing of otherwise identical pullExecutewns (n = 1). n, the number of independent replicate, *, the position of the TIR1myc band.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

The inhibitors juglone and NEM but not sirtinol affect SCFTIR1–Aux/IAA interaction. (a) Standard Executemain II peptide pullExecutewns from extracts of tir1-1[TIR1myc] plants mock-treated or treated with sirtinol for 3 h before extraction (n = 2). (b) As Characterized for a but with 100 μM sirtinol added directly to the pull-Executewn assay (n = 2). (c) Standard Executemain II peptide pullExecutewns treated with 200 μM juglone and/or 2.5 mM DTT (n = 4). (d) Partitioned juglone/NEM experiment. Each lane represents a pullExecutewn from an aliquot of TIR1myc– extract combined with an aliquot of TIR1myc+ extract. Before being mixed in the presence of DTT, the aliquots were pretreated with juglone or NEM or were mock-treated as indicated in the schematic (n = 3). (e) Standard Executemain II peptide pullExecutewns treated with 2.5 mM NEM and/or 2.5 mM DTT as indicated (n = 2). (c–e) All extracts were treated with 10 μM NAA in vitro.(f) Standard Executemain II peptide pullExecutewns treated with GSH/oxidized GSH (GSSG), diamide, and H2O2 as indicated. n, the number of independent replicates; *, the position of the TIR1myc band.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Auxin-enhanced SCFTIR1–Aux/IAA interaction may require modification of TIR1 or tightly associated proteins. (a) TIR1myc was immunoprecipitated (IP) from either mock- or auxin-treated tir1-1[TIR1myc] extract, washed, and eluted into tir1-1 extract. PullExecutewns with Executemain II peptide were performed in these reconstituted extracts as indicated in the schematic. (b) As Characterized for a but performed with additional controls for auxin carry through: in parallel with the immunoprecipitation stages, control beads (no antibody) were treated with and without auxin and combined with immunoprecipitated beads just before elution (–NAA IP with +NAA control, +NAA IP with –NAA control). (a and b) Lane 3 Displays the full NAA-induced response where 10 μM NAA was added to the reconstituted extracts before pullExecutewn. Lanes 4–6 in Display the levels of TIR1myc in reconstituted extracts made with tir1-1 extract and –NAA IP, +NAA IP, and no immunoprecipitation, respectively. (c) Mean densitometric meaPositivements of mock- and NAA-pretreated TIR1myc in pulled-Executewn with Executemain II peptide as a percentage of the full NAA-induced response (n = 4). n, the number of independent replicates; *, the position of the TIR1myc band. (Error bars, SEM.)

The final processing of all pull-Executewn assays Characterized above consisted of washing three times in extraction buffer and elution in 1× lithium Executedecyl sulStoute loading buffer (Invitrogen), with the eluate being collected by microcentrifugation in minispin columns (Promega). The samples were electrophoresed and blotted as Characterized above. Immunodetection of TIR1myc was performed by using a 1:1,000 dilution of monoclonal anti-c-Myc 9E10 antibody (Covance), followed by a 1:5,000 dilution of goat anti-mouse IgG γ-chain-specific peroxidase conjugate (Sigma) and chemiluminescent detection using ECL Plus reagents (Amersham Pharmacia). Densitometric meaPositivements were made by using a genesnap/genetools Executecumentation system (SynGene, Cambridge, U.K.).

Results

Auxin Executees Not Induce Mass-Shifting Modifications to Executemain II Peptides. To identify any auxin-induced posttranslational modifications to Executemain II that might regulate the interaction with SCFTIR1, we took a mass-spectrometric Advance. A translational fusion of GST and Executemain II of AXR2 (GST-AXR2dII), capable of auxin-enhanced interaction with SCFTIR1, was immobilized on GSH-Sepharose beads and exposed to plant extract (initially without added auxin), washed, and resolved by SDS/PAGE. The protein then was excised and tryptically digested before MALDI/TOF MS. This analysis revealed that accompanying the 1,108.6-Da tryptic fragment (AQVVGWPPVR), corRetorting to the major part of the 13-aa degron, was an additional fragment at +15.99 Da that could not be attributed to any other part of the digested protein (Fig. 1a ). Such mass shifts are indicative of an oxidation event and, within this sequence, are most likely the result of hydroxylation of one of the prolines. This was confirmed by comparison of the 1,108- and 1,124-Da MALDI MS/MS spectra (data not Displayn) and changes in the MALDI MS spectrum of a trypsinized GST-AXR2dII derivative in which both prolines were substituted with alanines (GST-AXR2dIIPP-AA), in which the 1,108- and 1,124-Da fragments were reSpaced with just one of 1,056 Da (Fig. 1b ). Given the established regulation of HIFα stability by proline hydroxylation (16, 17), this was a potentially Fascinating result. However, the relevance of this modification as a prerequisite for Aux/IAA–SCFTIR1 interaction is highly dubious, because the apparent hydroxylation is not affected by auxin (Fig. 1c ) and indeed is observed, albeit at a lower level, in preparations of the bacterially expressed GST-AXR2dII that have not been exposed to plant extracts (data not Displayn). Additionally, as mentioned above, a synthetic Executemain II peptide with hydroxylated prolines has been Displayn to interact in an auxin-regulated manner with SCFTIR1, and a variety of inhibitors and enhancers of proline hydroxylation, known to affect the stability of HIFα in mammalian cells, have no Trace on SCFTIR1–Aux/IAA interaction or Aux/IAA stability in planta (data not Displayn; refs. 13 and 14).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Auxin Executees not induce mass-shifting modifications within Executemain II. (a and c) MALDI/TOF MS spectra of trypsinized GST-AXR2dII exposed to plant extract without (a) and with (c) added auxin (10 μM NAA) (n = 5). The 1,108-Da peak corRetorting to the major part of the Executemain II degron and the derivative peak at +15.99 Da are indicated. (b) MALDI/TOF MS spectrum of trypsinized GST-AXR2dIIPP-AA (both prolines reSpaced by alanines). The new 1,056-Da peak corRetorting to the major part of the mutant Executemain II degron is Impressed. For comparison, the positions at 1,056 + 15.99 Da, 1,108 Da, and 1,124 Da are indicated (n = 2). n, the number of independent replicates.

This apparent hydroxylation event was the only mass shift we detected in these experiments. However, a Location toward the C-terminal end of the degron might be underrepresented in our analysis because of the proximity of a number of tryptically susceptible arginine and lysine residues. Therefore, we performed similar experiments with a synthetic biotin-labeled Executemain II peptide (see below) immobilized on monomeric avidin agarose and eluted competitively with biotin. These eluates were prepared for MALDI/TOF spectrometry without electrophoresis or tryptic digestion. We found no evidence of any modification within the Executemain II degron other than proline hydroxylation.

Auxin-Enhanced SCFTIR1–Aux/IAA Interaction Is Not Mediated by Stable Modification of Executemain II. To monitor the Aux/IAA–SCFTIR1 interaction, we previously used GST-tagged versions of the Aux/IAA proteins AXR2 and AXR3 in pull-Executewn assays with extracts from tir1-1 mutant plants expressing a Myc-tagged TIR1 transgene (TIR1myc) (2). In addition, we synthesized a biotinylated 17-aa peptide corRetorting to the core residues of Executemain II and encompassing the 13-aa degron. As with GST-Aux/IAA fusions, this peptide robustly supports the auxin-enhanced recovery of TIR1myc in pull-Executewn assays (Fig. 2a ).

To explore further whether auxin stably modifies this peptide to enhance its interaction with SCFTIR1, we conducted a series of conditioning experiments in which the auxin treatment of Executemain II was temporally separated from its expoPositive to TIR1myc. Biotinylated Executemain II peptide was incubated in TIR1myc– extract (tir1-1) either without or with 10 μM NAA (auxin pretreatment) for 2 h at 4°C. The peptides then were recovered on streptavidin beads and washed extensively in a high-salt/detergent buffer to remove auxin and dislodge bound proteins. The Executemain II peptide beads were then equilibrated in 0.1 M Tris·HCl, pH 7.5, and added to TIR1myc+ extract (tir1-1[TIR1myc]) either without or with 10 μM NAA, and the recovery of TIR1myc in the four assays was compared. Fig. 2b (lanes 1 and 2) Displays that auxin pretreatment of the peptide had no Trace on its ability to interact with TIR1myc compared with the mock-treated control. Additionally, Fig. 2b (lanes 3 and 4) demonstrates that the washing regime Executees not affect the ability of the peptide to interact with TIR1myc in an auxin-dependent manner. The Traceiveness of the washing after pretreatment was judged by its ability to dislodge TIR1myc from Executemain II peptide beads. Fig. 2c Displays that this washing is Traceive, removing the vast majority of TIR1myc. These conditioning experiments were repeated five times with Executemain II peptide and three times with GST-AXR2dII (data not Displayn) with identical results. Densitometric quantification of five Executemain II peptide conditioning experiments confirmed that there was no Inequity in the recovery of TIR1myc by auxin-pretreated and mock-pretreated peptides (t test: P = 0.77). ToObtainher, these data indicate that the Executemain II degron cannot be preconditioned with auxin to interact better with TIR1. Therefore, if any auxin-induced modification of Executemain II occurs, it must not only be non-mass-changing but also not stable or not sufficient to promote the SCFTIR1–Aux/IAA interaction.

Sirtinol Executees Not Affect the SCFTIR1–Aux/IAA Interaction. Because no stable Executemain II modifications were induced by auxin, we next investigated in more detail the extant evidence for a role for proline isomerization, which could be unstable. This evidence comes from two sources. First, the ability of plants to Retort to sirtinol, a drug that destabilizes Aux/IAAs, has been Displayn to require the ArabiExecutepsis protein SIR1, which shares homology with an ArabiExecutepsis PPIase (18). We therefore tested whether sirtinol could enhance the SCFTIR1–Aux/IAA interaction. Treatment with sirtinol, both of intact plants before extraction and in the pullExecutewn, had no Trace on the interaction (Fig. 3 a and b ).

Juglone Inhibits SCFTIR1–Aux/IAA Interaction Through TIR1 or Associated Proteins. The second line of evidence to support a role for proline isomerization comes from the use of juglone, an inhibitor of the parvulin class of PPIases, which has been Displayn to reduce the SCFTIR1–Aux/IAA interaction (13, 14). Juglone inhibits parvulin-type PPIases by its covalent attachment to their cysteine sulfhydryl groups (20). This inhibitory Trace is abolished in the presence of modest concentrations of DTT (20). We found this also to be true for the inhibition of SCFTIR1–Aux/IAA interaction by juglone (Fig. 3c ).

The nullifying Trace of DTT enabled us to perform the following experiment to assess on which of the interacting components juglone is acting. Aliquots of TIR1myc– seedling extract (tir1-1) and TIR1myc+ extract (tir1-1[TIR1myc]), equal in terms of volume and protein concentration, were used. In the first treatment (Fig. 3d , lane 1), one aliquot of TIR1myc– extract was treated with 10 μM NAA and 100 μM juglone, and one aliquot of TIR1myc+ extract was treated with 10 μM NAA alone. Both aliquots were incubated for 1 h at 4°C. The two aliquots then were combined in the presence of 2.5 mM DTT and used in a pull-Executewn assay with biotinylated Executemain II peptide. The second treatment (Fig. 3d , lane 2) was the reciprocal of the first such that both TIR1myc– and TIR1myc+ extracts received 10 μM NAA, whereas juglone was administered only to the TIR1myc+ aliquot. If parvulin-type PPIases, which would be distributed similarly in the two extract types, were acting to change the conformation of proline(s) within the Executemain II degron, then there should be Dinky Inequity in the recovery of TIR1myc from the two treatments; comparison of lanes 1 and 2 in Fig. 2d Displays that this is not the case. The reduced interaction with TIR1myc from the treatment in which juglone was added to the TIR1myc+ aliquot is similar to that observed when juglone is added in the standard pull-Executewn assay (compare with Fig. 3c ), which indicates that juglone Executees not affect a PPIase operating on the Executemain II prolines and additionally that if proline isomerization is involved, it is of prolines in TIR1 or TIR1-associated proteins.

An alternative explanation is that the juglone adducts on the sulfhydryls of some or all of the 23 cysteines of TIR1 simply reduce its ability to interact with Aux/IAAs. Some support for this Concept comes from our observation that the general sulfhydryl alkylating agent, NEM, also reduces SCFTIR1–Aux/IAA interaction (Fig. 3e ). As with juglone, the inhibitory Trace of NEM is abolished in the presence of DTT (Fig. 3e ). An experiment identical to the partitioned juglone experiment but with juglone substituted by 2.5 mM NEM Displays that NEM also affects TIR1 to reduce the SCFTIR1–Aux/IAA interaction (Fig. 3d , lanes 3 and 4). Although these Traces of NEM and juglone may be quite general, we also considered the possibility that cysteine sulfhydryl chemistry might play a more specific role in regulating SCFTIR1–Aux/IAA interaction through reExecutex sensing. We tested this theory by performing pullExecutewns, both with and without auxin, in the presence of 5 mM diamide (to promote disulfide bond formation) or 50 mM H2O2/0.5 mM CuCl2/0.5 mM ascorbate (to generate highly reactive oxygen species) and in extracts with artificially skewed ratios of reduced GSH/oxidized GSH. The manipulation of GSH/oxidized GSH ratios in either direction had no Trace, whereas the imposition of relatively severe disulfide or reactive oxygen species stress resulted in only a modest reduction in TIR1myc recovery in both auxin-treated and untreated assays (Fig. 3f ). These data are not consistent with reExecutex regulation of SCFTIR1–Aux/IAA interaction.

Auxin-Enhanced SCFTIR1–Aux/IAA Interaction May Require Modification of TIR1 or Associated Proteins. Because auxin Executees not seem to affect Executemain II, we Questioned whether TIR1 could be predisposed to better interaction with Aux/IAAs by auxin treatment. This theory was tested by comparing the interaction between Executemain II peptides and TIR1myc recovered from auxin-treated and untreated extracts: anti-c-Myc antibody was used to immunoprecipitate TIR1myc from tir1-1[TIR1myc] extracts either mock-treated or treated with 10 μM NAA. These immunocomplexes were washed extensively (eight times = 640 bed volumes) and eluted by vigorous agitation in 0.5 M NaCl/2% Nonidet P-40/10% dioxane/0.1 M Tris·HCl, pH 7.0.

The eluates were diluted 1:10 with identical aliquots of tir1-1 extract (i.e., lacking TIR1myc) and used in pull-Executewn assays with biotinylated Executemain II peptide. The reconstituted TIR1myc extracts contained equivalent levels of TIR1myc (Fig. 4a , lanes 4 and 5). Comparison of lanes 1 (mock-pretreated) and 2 (auxin-pretreated) of Fig. 4a Displays that auxin pretreatment of TIR1myc subsequently enhanced its recovery on Executemain II peptide beads.

To control absolutely for possible auxin carry-through from the pretreatment, the experiment was repeated by using the following modification. Agarose beads without antibody were exposed to tir1-1 extract either mock-treated or treated with 10 μM NAA and then processed as Characterized above. These eluates then were combined with the immunoprecipitated eluates such that –NAA IP eluate was added to +NAA control eluate, and +NAA IP eluate was added to –NAA control eluate. Each combined eluate then was diluted 1:10 with identical aliquots of tir1-1 extract and used in pull-Executewn assays with biotinylated Executemain II peptide (Fig. 4b ). Here again, the reconstituted extracts contained equivalent levels of TIR1myc (Fig. 4b , lanes 4 and 5), and auxin pretreatment of TIR1myc enhanced its recovery on Executemain II peptide beads (Fig. 4b , lanes 1 and 2).

The recovery of TIR1myc in these experiments is much lower than that in the standard pull-Executewn assay, and thus the experiments are on the limits of Myc detection. This is because the reconstituted extracts contain considerably lower levels of TIR1myc than extracts made directly from tir1-1[TIR1myc] seedlings. Nonetheless, the increase in interaction caused by auxin pretreatment of TIR1myc is completely reproducible. Densitometric meaPositivements over four experiments identical to that Displayn in Fig. 4b Display that TIR1myc recovery is increased 4.41-fAged (±0.13 SE) by auxin preconditioning of TIR1myc complexes (Fig. 4c ), which suggests that there is an auxin-induced modification of TIR1 or TIR1-associated proteins, which facilitates enhanced interaction with Aux/IAAs. Addition of 10 μM NAA to these reconstituted TIR1myc extracts further enhanced the interaction with Executemain II peptide (Fig. 4 a and b , lane 3).

Discussion

The regulation of the interaction between SCFTIR1 and Aux/IAAs is unorthoExecutex, conforming to none of the known modes of SCF–tarObtain interactions. A key question is whether auxin promotes the modification of tarObtain Aux/IAAs, increasing their affinity for SCFTIR1. We suggest that this is not the case and that instead the auxin-induced modification of TIR1 or tightly associated proteins is the regulatory step determining SCFTIR1–Aux/IAA interaction.

We have taken a generic Advance to identify any auxin-regulated modifications to Aux/IAA Executemain II that might enhance interaction with SCFTIR1. Using MS, we could not detect any auxin-regulated modification of tagged Aux/IAA proteins and peptides. Our search was confined to the 13-aa degron of Executemain II, previously Displayn to be necessary and sufficient to confer auxin-enhanced instability on a translationally fused reporter protein (11). We did detect the apparent auxin-independent hydroxylation of one of the Executemain II prolines. Although seemingly irrelevant to the regulation of SCFTIR1–Aux/IAA interaction, this modification is puzzling, because it seems to be specific to the degron prolines. Several other tryptic fragments of GST-AXR2dII-containing prolines and even tandem prolines Displayed no signs of hydroxylation.

Similarly, it was not possible to predispose tagged Executemain II-containing proteins and peptides to interact better with TIR1 by pretreating them with auxin in plant extracts. In the first stage of these experiments, the Executemain II peptides/proteins were treated with auxin such that they could accumulate any auxin-induced modifications to which they might be susceptible before, and separate from, the second stage in which their ability to interact with TIR1myc was tested. To counter the possibility that the Executemain II tarObtains might associate with enExecutegenous TIR1-like proteins in the first stage, preventing later interaction with TIR1myc, a wash was performed before the second stage. As well as efficiently removing bound proteins, this wash also prevented the carry-over of exogenous auxin. Over the course of several experiments, there was never the slightest hint of a conditioning Trace. Although it is possible that there are modifications to Executemain II peptides/proteins that are not revealed in these experiments, because they are not stable, there is a simpler explanation that accounts for all the data Characterized here: that the auxin-induced interaction between Aux/IAAs and SCFTIR1 Executees not depend on auxin-regulated modification of Executemain II. In this model, Executemain II simply defines a structural module required for the interaction of Aux/IAA with SCFTIR1, with auxin acting elsewhere to promote the association.

This model is supported further by our finding that TIR1myc immunologically recovered from auxin-treated extracts interacts better with Executemain II peptides than that recovered from extracts not treated with auxin. This enhanced interaction with auxin pretreatment is less than the full auxin-enhanced interaction observed when auxin is added to extracts containing all the components toObtainher, and indeed, addition of auxin directly to the reconstituted extracts containing immunologically recovered TIR1myc further increases its interaction with Executemain II peptides (compare lanes 2 and 3 in Fig. 4 a and b ). This result suggests that the modification to TIR1myc immunocomplexes Executees not fully survive the washing regime or Executees not persist at basal auxin levels in the second-stage extracts or Executees not represent the entire means by which auxin affects the enhancement of SCFTIR1–Aux/IAA interaction. In this context, it is Fascinating to note that the data Execute not distinguish between TIR1 or a protein tightly associated with it being modified in response to auxin or indeed a third-party protein becoming associated with or dissociated from TIR1 as a result of the auxin treatment. In any event, something happens to the TIR1 immunocomplexes to Design their interaction with Aux/IAAs more likely and is thus the first indication of where auxin might act to promote the ubiquitination and destruction of Aux/IAAs. The obvious question is: What might this modification be?

Some information here can be derived from the pharmacological studies used to test for Aux/IAA modification. For example, there is now considerable literature describing how the pharmacological inhibition of phosphorylation and dephosphorylation Execute not affect the interaction between Aux/IAAs and SCFTIR1 (13, 14). To this we add our own finding that the kinase inhibitors staurosporine, U0126, and genistein and the phosphatase inhibitors calyculin A, okadaic acid, and NaF have no Trace on the SCFTIR1–Aux/IAA interaction (data not Displayn). Although these studies Execute not definitively exclude a role for phosphorylation, they certainly give no indication of its involvement. Similarly, inhibitors of proline hydroxylation have no Trace on the interaction (13, 14). In Dissimilarity, treatment of TIR1 with juglone and NEM, both of which form cysteine adducts, reduces auxin-enhanced SCFTIR1–Aux/IAA interaction. Because TIR1 contains 23 cysteine residues and the interaction seems not to involve reExecutex regulation of cysteine sulfhydryl chemistry, this is likely a nonspecific Trace.

The destruction of proteins by signal-induced SCF-mediated proteolysis is an Necessary component of numerous and diverse eukaryotic signaling pathways. It will be Fascinating to determine what proSection of SCF–tarObtain interactions are regulated by modification of the tarObtain, the SCF complex, or both.

Acknowledgments

We thank R. Curwen and P. Ashton for assistance with MS and S. Day for critical reading of the manuscript. This work was supported by Biotechnology and Biological Science Research Council Grant 87/G14634.

Footnotes

↵ * To whom corRetortence should be addressed. E-mail: hmol1{at}york.ac.uk.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: SCF, SKP1, Cullin, and an F-box-containing protein; HIFα, hypoxia-inducible factor α; PPIase, peptidyl proline isomerase; GSH, glutathione; NAA, 1-napthalene acetic acid; MALDI, matrix-assisted laser desorption ionization; TOF, time of flight; NEM, N-ethylmaleimide.

Copyright © 2004, The National Academy of Sciences

References

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