Abstract
The Arabidopsis thaliana genome encodes several families of polypeptides that are known or predicted to participate in the formation of the SCF-class of E3-ubiquitin ligase complexes. One such gene family encodes the Skp1-like class of polypeptide subunits, where 21 genes have been identified and are known to be expressed in Arabidopsis. Phylogenetic analysis based on deduced polypeptide sequence organizes the family of ASK proteins into 7 clades. The complexity of the ASK gene family, together with the close structural similarity among its members raises the prospect of significant functional redundancy among select paralogs. We have assessed the potential for functional redundancy within the ASK gene family by analyzing an expanded set of criteria that define redundancy with higher resolution. The criteria used include quantitative expression of locus-specific transcripts using qRT-PCR, assessment of the sub-cellular localization of individual ASK:YFP auto-fluorescent fusion proteins expressed in vivo as well as the in planta assessment of individual ASK-F-Box protein interactions using bimolecular fluorescent complementation techniques in combination with confocal imagery in live cells. The results indicate significant functional divergence of steady state transcript abundance and protein-protein interaction specificity involving ASK proteins in a pattern that is poorly predicted by sequence-based phylogeny. The information emerging from this and related studies will prove important for defining the functional intersection of expression, localization and gene product interaction that better predicts the formation of discrete SCF complexes, as a prelude to investigating their molecular mode of action.
Highlights
Genetic and molecular studies in the model plant species Arabidopsis thaliana have emphasized the importance of ubiquitinmediated targeted protein degradation for the regulation of diverse plant-specific processes [1,2,3]
This clustering of the predicted ASK proteins is consistent with published results, where ASK1 and ASK2 are grouped within a clade and were found to overlap functionally and are known to be essential for early development in Arabidopsis [40]
We used this phylogenetic tree as a basis to assess the functional relatedness among ASK paralogs as measured by three functional criteria: tissue/organ-specific transcript abundance, sub-cellular localization of yellow fluorescent protein (YFP)-tagged ASK proteins, and the protein-protein interaction profiles of ASK proteins in conjunction with selected F-Box proteins expressed as bimolecular fluorescence complementation (BiFC) fusion constructs
Summary
Genetic and molecular studies in the model plant species Arabidopsis thaliana have emphasized the importance of ubiquitinmediated targeted protein degradation for the regulation of diverse plant-specific processes [1,2,3]. 27,000 protein coding genes, it has been annotated to contain over 1,500 genes that are known or predicted to encode subunits of ubiquitin ligase complexes (nearly 6% of the coding capacity) including more than 700 F-box proteins comprising about 3% of the Arabidopsis genome coding capacity [1]. This genetic complexity and allocation of gene coding capacity to SCFligase complexes involved in post-translational protein turnoverrelated processes is prominent in plants, and can be compared with that of Homo sapiens where only 69 F-Box genes have been identified [11]
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