Protein interaction analysis of SCF ubiquitin E3 ligase subunits from Arabidopsis

Abstract

SummaryUbiquitin E3 ligases are a diverse family of protein complexes that mediate the ubiquitination and subsequent proteolytic turnover of proteins in a highly speci®c manner. Among the several classes of ubiquitin E3 ligases, the Skp1-Cullin-F-box (SCF) class is generally comprised of three`core' subunits: Skp1 and Cullin, plus at least one F-box protein (FBP) subunit that imparts speci®city for the ubiquitination of selected target proteins. Recent genetic and biochemical evidence in Arabidopsis thaliana suggests that post-translational turnover of proteins mediated by SCF complexes is important for the regulation of diverse developmental and environmental response pathways. In this report, we extend upon a previous annotation of the Arabidopsis Skp1-like (ASK) and FBP gene families to include the Cullin family of proteins. Analysis of the protein interaction pro®les involving the products of all three gene families suggests a functional distinction between ASK proteins in that selected members of the protein family interact generally while others interact more speci®cally with members of the F-box protein family. Analysis of the interaction of Cullins with FBPs indicates that CUL1 and CUL2, but not CUL3A, persist as components of selected SCF complexes, suggesting some degree of functional specialization for these proteins. Yeast two-hybrid analyses also revealed binary protein interactions between selected members of the FBP family in Arabidopsis. These and related results are discussed in terms of their implications for subunit composition, stoichiometry and functional diversity of SCF complexes in Arabidopsis.

Arabidopsis Kelch Repeat F-Box Proteins Regulate Phenylpropanoid Biosynthesis via Controlling the Turnover of Phenylalanine Ammonia-Lyase

“…The cytokinin signaling cascade has a central role in regulating cell division; it also modulates the development of the shoots and roots and stress responses (Sakakibara, 2006). F-box proteins can selectively target a set of substrates (Craig and Tyers, 1999;del Pozo and Estelle, 2000;Gagne et al, 2002;Risseeuw et al, 2003;Ho et al, 2008). The alteration of phenylpropanoid-lignin biosynthesis, along with the disturbance of KFB gene expression, is unlikely attributable to the changes in Arabidopsis response regulator stability and the cytokinin response, since, for example, the reduction of the levels of phenolics and lignin in the KFB overexpression lines is not due to the downregulation of PAL transcription but to the decrease in enzyme concentration (Figures 10 and 11).…”

Ralstonia solanacearum requires F-box-like domain-containing type III effectors to promote disease on several host plants

“…When GST-GALA6⌬F-box was used, the interaction with ASK2 was no longer observed. (24,25). We systematically tested the interaction between the six cloned GALAs and the different ASKs by Y2H.…”

Characterization of theVIER F-BOX PROTEINEGenes fromArabidopsisReveals Their Importance for Plant Growth and Development

“…Interacts with the SCF Complex Adaptor Protein ASK2 and Localizes to the Cytoplasm F-box proteins interact via their F-box domain with the evolutionarily conserved SKP1 adaptor protein, which in turn binds to the Cullin1 subunit of SCF complexes (Deshaies, 1999;Zheng et al, 2002;Risseeuw et al, 2003). To show that the VFB proteins have retained the ability to bind SKP1 proteins, we used VFB2 as a representative family member to examine its interaction with ARABIDOPSIS SKP1-2 (ASK2), a predominant member of the Arabidopsis SKP1 protein family, using the yeast two-hybrid system (Risseeuw et al, 2003;Zhao et al, 2003).…”

“…To show that the VFB proteins have retained the ability to bind SKP1 proteins, we used VFB2 as a representative family member to examine its interaction with ARABIDOPSIS SKP1-2 (ASK2), a predominant member of the Arabidopsis SKP1 protein family, using the yeast two-hybrid system (Risseeuw et al, 2003;Zhao et al, 2003). Our analysis revealed an interaction between VFB2 and ASK2 but not between VFB2 and the two other SCF complex components RBX1 and CUL1 (Figures 2A and 2B) Risseeuw et al, 2003). As expected, a VFB2 deletion variant lacking the F-box domain (VFB2DF-box) failed to interact with ASK2 in the yeast assay (Figures 2A and 2B).…”

“…The fragments were introduced as Xho1 fragments into pEG202. The ASK2, RBX1, and CUL1 prey constructs were described previously (Schwechheimer et al, 2001;Risseeuw et al, 2003). Two-hybrid interactions were performed as described by .…”