Protein–protein interactions among the Aux/IAA proteins

Abstract

The plant hormone indoleacetic acid (IAA) transcriptionally activates early genes in plants. The Aux͞IAA family of early genes encodes proteins that are short-lived and nuclear-localized. They also contain a putative prokaryotic ␤␣␣ DNA binding motif whose formation requires protein dimerization. Here, we show that the pea PS-IAA4 and Arabidopsis IAA1 and IAA2 proteins perform homo-and heterotypic interactions in yeast using the two-hybrid system. Gel-filtration chromatography and chemical cross-linking experiments demonstrate that the PS-IAA4 and IAA1 proteins interact to form homodimers in vitro. Deletion analysis of PS-IAA4 indicates that the ␤␣␣ containing acidic C terminus of the protein is necessary for homotypic interactions in the yeast two-hybrid system. Screening an Arabidopsis -ACT cDNA library using IAA1 as a bait reveals heterotypic interactions of IAA1 with known and newly discovered members of the Arabidopsis Aux͞IAA gene family. The new member IAA24 has similarity to ARF1, a transcription factor that binds to an auxin response element. Combinatorial interactions among the various members of the Aux͞IAA gene family may regulate a variety of late genes as well as serve as autoregulators of early auxin-regulated gene expression. These interactions provide a molecular basis for the developmental and tissue-specific manner of auxin action.

Biochemical characterization of recombinant polypeptides correspondingto the predicted ²±± fold in Aux/IAA proteins

“…cently, it has been demonstrated that Aux/IAA proteins homo-and heterodimerize in vitro and in vivo [12]. Moreover, Aux/IAA proteins interact with members of the A R F protein family of transcription factors that contain C-terminal, Aux/ IAA-like domains III and IV [9,12].…”

“…Moreover, Aux/IAA proteins interact with members of the A R F protein family of transcription factors that contain C-terminal, Aux/ IAA-like domains III and IV [9,12]. Using a yeast two-hybrid system, deletion analysis of the PS-IAA4 protein from pea revealed that the C-terminus of PS-IAA4, containing domains III and IV, is necessary and sufficient for dimerization [12].…”

“…Moreover, Aux/IAA proteins interact with members of the A R F protein family of transcription factors that contain C-terminal, Aux/ IAA-like domains III and IV [9,12]. Using a yeast two-hybrid system, deletion analysis of the PS-IAA4 protein from pea revealed that the C-terminus of PS-IAA4, containing domains III and IV, is necessary and sufficient for dimerization [12]. However, due to the instability of some of the tested PS-IAA4 deletion proteins in yeast, the in vivo data were inconclusive with respect to the role of the predicted [3c~c~ fold of domain III in protein dimerization [12].…”

“…As indicated by the isolation of at least 20 IAA genes in Arabidopsis thaliana, large multigene families encode Aux/IAA proteins in plants [11,12]. Biochemical, molecular, and genetic studies suggest that Aux!…”

“…Interestingly, the C-terminal regions of most ARF proteins contain Aux/IAA-like conserved domains III and IV, which compose the C-terminus of Aux/IAA proteins [9,10]. These C-terminal regions mediate both intra-and interfamily protein-protein interactions of Aux/IAA and ARF family members [9,12]. Therefore, the predicted amphipathic ~c~c~ fold of conserved domain III in Aux/IAA and ARF proteins likely plays an important role in mediating such homo-and heterotypic interactions.…”