The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.ARM repeats are short 42-amino acid motifs that were first identified in the fruitfly (Drosophila melanogaster) segment polarity protein, armadillo (Riggleman et al., 1989). ARM repeats have been subsequently identified in a wide range of eukaryotic proteins, and these proteins interact with numerous other proteins via their ARM repeats, resulting in the regulation of a variety of cellular processes (for review, see Hatzfeld, 1999). Based on the crystal structure of the mammalian armadillo homolog, ␤-catenin, each ARM repeat forms a trihelical structure that folds into a superhelix, and six ARM repeats are proposed to constitute a protein interaction domain (Huber et al., 1997). In animals, wellcharacterized ARM repeat proteins include ␤-catenin/armadillo involved in the Wnt/wingless signaling pathway and cadherin-mediated cell adhesion, the APC tumor suppressor protein in Wnt signaling, and several other cadherin-associated ARM repeat proteins (Hatzfeld, 1999). In addition, there is the conserved nuclear import pathway in yeast (Saccharomyces cerevisiae), plants, and animals that involves the ARM repeat protein, Importin-␣, and the Importin-␤ protein with related HEAT repeats (Andrade et al., 2001).More recently, a new class of ARM repeat proteins was identified in plants where the ARM repeat region is preceded by a E3 ubiquitin ligase motif called the U-box (Amador et al., 2001; Azevedo et al., 2001;Stone et al., 2003). The ubiquitination of proteins involves three enzymes: the E1 ubiquitin-activating enzyme, which forms a thioester intermediate with ubiquitin; the E2 ubiquitin-conjugating enzyme, which receives the ubiquitin molecule from the E1 enzyme; and the E3 ubiquitin ligase, which facilitates t...

Yeast two-hybrid technology is used to build a high-quality protein interaction network centered on Arabidopsis G-protein coupled signaling. The interactions uncovered are without precedent in animals and fungi and help identify new cellular roles for G-protein signaling in plants.

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.

Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two Gbg dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single b (AGB1) or the two g (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/ AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops.