Auxin-enhanced SCF^TIR1–Aux/IAA interaction is not mediated by stable modification of the Aux/IAA. (a) A synthetic Aux/IAA domain II peptide was used in pull-down assays from extracts of tir1-1[TIR1myc] plants without and with auxin (10 μM NAA) added in vitro. The recovery of TIR1myc on domain II peptide beads was assessed by immunoblotting with anti-c-Myc antibody (n = 40). (b) Immobilized domain II peptides were mock-treated or auxin-treated in TIR1myc^– extract before being washed and used in pull-down 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 effect of high-salt/detergent washing of otherwise identical pulldowns (n = 1). n, the number of independent replicate, *, the position of the TIR1myc band.

Auxin-enhanced SCF^TIR1–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. Pulldowns with domain II peptide were performed in these reconstituted extracts as indicated in the schematic. (b) As described 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 shows the full NAA-induced response where 10 μM NAA was added to the reconstituted extracts before pulldown. Lanes 4–6 in show the levels of TIR1myc in reconstituted extracts made with tir1-1 extract and –NAA IP, +NAA IP, and no immunoprecipitation, respectively. (c) Mean densitometric measurements of mock- and NAA-pretreated TIR1myc in pulled-down with domain 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 inhibitors juglone and NEM but not sirtinol affect SCF^TIR1–Aux/IAA interaction. (a) Standard domain II peptide pulldowns from extracts of tir1-1[TIR1myc] plants mock-treated or treated with sirtinol for 3 h before extraction (n = 2). (b) As described for a but with 100 μM sirtinol added directly to the pull-down assay (n = 2). (c) Standard domain II peptide pulldowns treated with 200 μM juglone and/or 2.5 mM DTT (n = 4). (d) Partitioned juglone/NEM experiment. Each lane represents a pulldown 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 domain II peptide pulldowns 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 domain II peptide pulldowns treated with GSH/oxidized GSH (GSSG), diamide, and H₂O₂ as indicated. n, the number of independent replicates; *, the position of the TIR1myc band.

Auxin does not induce mass-shifting modifications within domain 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 corresponding to the major part of the domain II degron and the derivative peak at +15.99 Da are indicated. (b) MALDI/TOF MS spectrum of trypsinized GST-AXR2dIIPP-AA (both prolines replaced by alanines). The new 1,056-Da peak corresponding to the major part of the mutant domain II degron is marked. 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.