Protein-protein interactions among the Aux/IAA proteins

Abstract

The plant hormone indoleacetic acid (IAA) transcriptionally activates early genes in plants. The Aux/IAA family of early genes encodes proteins that are short-lived and nuclear-localized. They also contain a putative prokaryotic betaalphaalpha 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 betaalphaalpha containing acidic C terminus of the protein is necessary for homotypic interactions in the yeast two-hybrid system. Screening an Arabidopsis lambda-ACT cDNA library using IAA1 as a bait reveals heterotypic interactions of IAA1 with known and newly discovered members of the Arabidopsis Aux/IAA 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 Aux/IAA 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.

Figure 1

Homotypic interactions of IAA1, IAA2, and PS-IAA4 in yeast. (I) Growth of yeast transformants on SC (A and B), SC-His containing 25 mM 3-AT (C and D), and the colony color of the transformants determined by the filter-lift assay (E and F). A, C, and E show the homotypic interaction of IAA1. B, D, and F show the homotypic interactions of IAA2 and PS-IAA4. The numbers shown on A and B correspond to the numbers on the left column of II. O indicates the orientation of each plate. (II) The relative growth, colony color, and β-gal activity of the yeast transformed with the indicated plasmids. Growth on SC-His plates containing 25 and 100 mM 3-AT, respectively, was scored as follows: +++, strong growth; ++, good growth; +, poor growth; +/−, very little growth; −, no growth. The color of the colonies was determined by the filter-lift assay, and β-gal activity was quantified enzymatically (see Material and Methods). The enzyme activities are the average of three independent yeast colonies.

Figure 2

Homodimerization of IAA1 and PS-IAA4 in vitro. (A) Elution positions (○) of (His)₆-tagged IAA1 and (His)₆-tagged PS-IAA4 after gel filtration relative to those of the molecular weight standard (•). Ve/Vo, Elution volume/void volume. The x axis is in log scale. (B) Cross-linking of (His)₆-tagged IAA1 and (His)₆-tagged PS-IAA4. Ni-NTA purified (His)₆-tagged IAA1 (lanes 1 and 2) and (His)₆-tagged PS-IAA4 (lanes 3 and 4) were incubated in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of BS³ for 1 min at 25°C. After terminating the reactions, the proteins were run on SDS/PAGE gel, transferred to membrane, and immunodetected using a (His)₆ tag-specific mAb. Numbers on the right of the blot indicate the size of the molecular weight standards. (C) Cross-linking of ribonuclease A. Ribonuclease A, a monomeric protein, was incubated with (lane 2) or without (lane 1) BS³, run on SDS/PAGE gel, and stained with Coomassie blue. The arrow indicates the position of ribonuclease A (molecular weight, 13 kDa).

Figure 3

Interaction domain analysis of PS-IAA4 in the yeast two-hybrid system. (I) Schematic diagram showing the deletion constructs of PS-IAA4 (constructs 1–6). The numbers indicate the positions of the deletions (constructs 2–6) with regard to full-length PS-IAA4 (construct 1). The DNA sequences encoding the different peptides were fused in-frame to the DNA binding domain and transactivation domain of Gal4, respectively, and the constructs were transformed into Y190. Cells were grown on SC-His plates containing 25 and 100 mM 3-AT, respectively, and assayed for colony color and β-gal activity. (II and III) The tables show the relative growth, colony color, and β-gal activity of yeast cells containing the indicated plasmids.

Figure 4

Heterotypic interaction of IAA1 with IAA2 and PS-IAA4. (I) Growth of yeast cells transformed with the indicated plasmids on SC plates (A), SC-His plates containing 25 and 100 mM 3-AT, respectively (B), and the colony color of the transformants determined by filter-lift assay (C). The numbers shown on A correspond to the numbers of the plasmids shown on the left column of II in this figure and in Fig. 1. (II) The table shows the relative growth, colony color, and β-gal activity of the yeast cells transformed with the indicated plasmids.

Figure 5

(A) Sequence alignment and domain structure of Aux/IAA proteins isolated by two-hybrid screening compared with those of ARF1, ARF1-BP, and ETT/ARF3 (37, 38). Identical and conserved amino acid residues (at least 50% matches) are shaded (light brown) and appear in the consensus (capital and small letters, respectively). Conserved domains are underlined and indicated by Roman numerals. Amino acid residues conserved within a distinct lineage are colored. Amino acid residues that may form hydrophobic surfaces in the predicted conserved amphipathic βαα motif are indicated by (•). NLS indicates the nuclear localization signal. The numbers at the right of the sequences indicate the number of amino acid residues for the corresponding coding region of the cDNA. The consensus sequence shown at the top of the alignment was obtained from the IAA1–15 proteins (34). IAA1, the prototype Aux/IAA protein, is shown below the consensus. (B) Similarity of the N-terminal region of IAA24, ARF1, ARF1-BP, and ETT/ARF3 with a portion of the C-terminal region of VP1 (39) and ABI3 (40).