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 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.
Ramos
J A
JA
Biochemistry and Molecular Biology Graduate Group, Section of Molecular and Cellular Biology, University of California, 1 Shields Avenue, Davis, California 95616, USA.
Zenser
N
N
Leyser
O
O
Callis
J
J
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D007210:Indoleacetic Acids / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011523:Protoplasts / Q000378:metabolism; D016415:Sequence Alignment; D014026:Nicotiana / Q000378:metabolism*
D007210:Indoleacetic Acids; D009097:Multienzyme Complexes; D010940:Plant Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex