Figure 2. SCF^TIR1-dependent and specific IAA6 and IAA19 ubiquitylation is enhanced by auxin.

(a) IVU assays with recombinant GST-IAA6 or GST-IAA19, E1 (AtUBA1), E2 (AtUBC8), reconstituted SCF^TIR1 (AtSKP1-TIR1, HsCul1 and MmRBX1), AtUb and IAA (auxin). AUX/IAA ubiquitylation is dependent on each component. Anti-GST, as well as anti-Ub antibodies recognize IAA6 and IAA19 ubiquitylated species. IVU reaction time 10 min (b) UBC8 and UBC1 Ub-conjugating enzymes (E2s) elicit poly-Ub-conjugation of IAA6 and IAA19 after 10 min in vitro. UBC8 promotes various Ub-linkages as seen by the reduction of IAA6 and IAA19 ubiquitylated species using either one of the chain-specific Ub-donors, Lys-29, Lys-48 or Lys-63 (Supplementary Fig. 9). (c) Rapid IVU of IAA6 and IAA19 is auxin- and time-dependent. Time course IVU reactions were performed using fluorescein isothiocyanate-labelled ubiquitin with or without 750 nM IAA. Ubiquitylation was monitored using the ubiquitin fluorescent signal (green, top) and Alexa Fluor 647-conjugated secondary antibodies for detection of GST:AUX/IAAs (magenta, middle). (*) Asterisk depicts ubiquitylated Cullin1 as previously reported⁷⁹. ImageQuant TL software was used for quantification and generation of merged image (bottom). Ratios for auxin- and target-dependent ubiquitylation were calculated from three independent IVUs (n=3; see Supplementary Fig. 8b–d for replicates) and the single measurements are depicted in the corresponding scatter dot plots with line at median. A two-way ANOVA (P>0.05) showed no significant differences for the relative Ub signal between IAA6 and IAA19 in a specific time point (left), or for the IAA19/IAA6 ratio with or without 750 nM IAA. (d) Increased nanomolar concentrations of IAA promote IAA6 or IAA19 ubiquitylation after 10 min IVU reactions and higher molecular ubiquitylated species occurred on IAA19.