Rapid degradation of auxin/indoleacetic acid proteins requires conserved amino acids of domain II and is proteasome dependent

Auxin rapidly induces auxin/indoleacetic acid (Aux/IAA) transcription. The proteins encoded are short-lived nucleus-localized transcriptional regulato rs that share four conserved domains. In a transient assay measuring protei n accumulation, an Aux/IAA 13-amino acid domain II consensus sequence was s ufficient to target firefly luciferase (LUC) for low protein accumulation e quivalent to that observed previously for full-length PSIAA6. Single amino acid substitutions in these 13 amino acids, corresponding to known auxin re sponse mutants, resulted in a sixfold to 20-fold increase in protein accumu lation. Naturally occurring variant amino acids had no effect. Residues ide ntified as essential by single alanine substitutions were not sufficient wh en all flanking amino acids were alanine, indicating the importance of flan king regions. Using direct protein degradation measurements in transgenic A rabidopsis seedlings, full-length IAA1, PSIAA6, and the N-terminal 73 PSIAA 6 amino acids targeted LUC for rapid degradation with 8-min half-lives. The C-terminal 109 amino acids did not affect LUC half-life. Smaller regions c ontaining domain If also targeted LUC for rapid degradation, but the rates were not equivalent to those of the full-length protein. A single domain II I substitution in the context of full-length PSIAA6 increased half-life 30- fold. Proteasome inhibitors affected Aux/IAA::LUC fusion protein accumulati on, demonstrating the involvement of the proteasome.