Fig. 3: Auxin-driven and SCFTIR1-dependent ubiquitylation of IAA7 and IAA12 display distinct dynamics. | Nature Communications

Fig. 3: Auxin-driven and SCFTIR1-dependent ubiquitylation of IAA7 and IAA12 display distinct dynamics.

From: Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies

Fig. 3

a IVU assays with recombinant GST-IAA7 or GST-IAA12, E1 (AtUBA1), E2 (AtUBC8), reconstituted SCFTIR1 (AtSKP1·TIR1, HsCul1 and MmRBX1), fluorescein-labeled ubiquitin (Ub) and IAA (auxin). IAA7 and IAA12 ubiquitylation is auxin-driven and time-dependent. Ubiquitylation was monitored using the ubiquitin fluorescent signal (green), and anti-GST/Alexa Fluor 647-conjugated antibodies for detection of GST-AUX/IAAs (magenta). ImageQuantTL software was used for quantification (middle; means ± SEM, n = 3), and generation of merged image (bottom; overlapping Ub and GST signal: yellow). b IAA7 and IAA12 IVU samples were analyzed via LC-MS, and putative ubiquitylation sites, detected by the diGly (or LRGG) Ub remnant after tryptic digest, were mapped relative to the domain structure. IAA12 Ub sites agglomerate in the region upstream of the degron (white) and the degron tail (light pink). c Ubiquitin conjugation on chimeric IAA7 and IAA12 (colors as in a) proteins in the presence or absence of 1 µM IAA. IVU reaction time 1 h. (*) Asterisks depict unmodified AUX/IAAs.

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