Rapid Degradation of Auxin/Indoleacetic Acid Proteins Requires Conserved Amino Acids of Domain II and Is Proteasome Dependent

Abstract

Auxin rapidly induces auxin/indoleacetic acid (Aux/IAA) transcription. The proteins encoded are short-lived nucleus-localized transcriptional regulators that share four conserved domains. In a transient assay measuring protein accumulation, an Aux/IAA 13-amino acid domain II consensus sequence was sufficient to target firefly luciferase (LUC) for low protein accumulation equivalent to that observed previously for full-length PSIAA6. Single amino acid substitutions in these 13 amino acids, corresponding to known auxin response mutants, resulted in a sixfold to 20-fold increase in protein accumulation. Naturally occurring variant amino acids had no effect. Residues identified as essential by single alanine substitutions were not sufficient when all flanking amino acids were alanine, indicating the importance of flanking regions. Using direct protein degradation measurements in transgenic Arabidopsis seedlings, full-length IAA1, PSIAA6, and the N-terminal 73 PSIAA6 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 containing domain II also targeted LUC for rapid degradation, but the rates were not equivalent to those of the full-length protein. A single domain II substitution in the context of full-length PSIAA6 increased half-life 30-fold. Proteasome inhibitors affected Aux/IAA::LUC fusion protein accumulation, demonstrating the involvement of the proteasome.

Auxin-induced SCF TIR1 –Aux/IAA interaction involves stable modification of the SCF TIR1 complex

“…Using MS, we could not detect any auxin-regulated modification of tagged Aux͞IAA proteins and peptides. Our search was confined to the 13-aa degron of domain II, previously shown 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 domain II prolines.…”

“…Most of the characterized canonical SCF-target protein interactions from yeast and mammals depend on the phosphorylation of the target protein (8). However, several lines of evidence suggest that phosphorylation does not play a role in regulating the SCF TIR1 -Aux͞IAA interaction: The 13-aa degron, necessary and sufficient for auxin-induced destabilization of Aux͞IAAs, does not contain any essential phosphorylatable residues (11), and a broad spectrum of kinase and phosphatase inhibitors does not interfere with the interaction (13, 14). There are a few known exceptions to the phosphorylation paradigm.…”

“…In the case of SCF TIR1 , the TIR1 F-box protein contains leucine-rich repeats (9), which are required to destabilize the Aux͞IAAs (2). Domain II of the Aux͞IAA is the site of the destabilization signal required for interaction with SCF TIR1 (10)(11)(12). A 13-aa core region of domain II has been shown to be necessary and sufficient to confer auxin-regulated SCF TIR1 interaction and instability on translationally fused reporter proteins and hence constitutes a degron (11).…”

“…Domain II of the Aux͞IAA is the site of the destabilization signal required for interaction with SCF TIR1 (10)(11)(12). A 13-aa core region of domain II has been shown to be necessary and sufficient to confer auxin-regulated SCF TIR1 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 SCF TIR1 (11).…”

“…A 13-aa core region of domain II has been shown to be necessary and sufficient to confer auxin-regulated SCF TIR1 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 SCF TIR1 (11). Mutations in this core region result in increased and auxin-resistant Aux͞IAA stability, reduced SCF TIR1 interaction, and, in the context of an otherwise intact Aux͞IAA in planta, a plethora of auxin-response phenotypes (2, 7, 13).…”