Characterization of Novel Shewanella- and Rhizobiales-Like PPP-Family Protein Phosphatases from Arabidopsis thaliana
Date
2013-07-10
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Abstract
Reversible protein phosphorylation catalyzed by protein kinases and phosphatases represents the most prolific and currently best-characterized post-translational modification. Over the last decade advancements in genome sequencing technologies has massively increased genomic
databases, resulting in the identification of previously unannotated protein kinases and
phosphatases from multiple organisms. The primary goal of the research presented here was to elucidate the evolutionary, biochemical, cellular and biological characteristics of two recently identified PPP-family protein phosphatase subclasses from the model photosynthetic Eukaryote
Arabidopsis thaliana. These two subclasses included the Shewanella-like (SLP1 and 2) and
Rhizobiales-like (RLPH2) phosphatases, which were named after their relatedness to phosphatase orthologs from Shewanella and Rhizobiales bacteria. Heterologous protein
phosphatase expression in, and purification from, Escherichia coli revealed unique biochemical characteristics including a complete insensitivity to PPP-family protein phosphatase inhibitors okadaic acid and microcystin-LR, as well as diversity in their phosphorylated substrate specificities. Bioinformatics complemented cell biology also uncovered unique subcellular localizations for each enzyme, with AtSLP1, 2 and AtRLPH2 being chloroplastic, mitochondrial and nuclear / cytosolic, respectively. Further identification of the AtSLP1 and AtSLP2 protein interactome from both plant tissues and cell culture was accomplished by employing a tandem affinity purification protein isolation strategy, with specific protein interactor complements indicating independent regulatory roles within plant cells involving chloroplast energy
biosynthesis and mitochondrial intermembrane space processes, respectively. Lastly, phenotypic analysis of atslp2 insertional mutant and 35S::AtSLP2 over-expression plants revealed biological involvement in regulating gibberellic acid-related processes during seed germination as well as influencing fatty acid and amino acid contents during seed maturation. Research presented here has made significant progress in our understanding of the previously uncharacterized SLP and RLPH PPP-family phosphatase subclasses from Arabidopsis thaliana, by resolving their molecular evolution, in addition to their subcellular, cellular and biochemical properties. A
functional understanding of AtSLP1 and AtSLP2 was also achieved through the identification of their protein interaction partners, which, in conjunction with insertional knockout and protein over-expression plant lines, lays a solid foundation for future research endeavors looking to examine bacterial-like phosphatases from photosynthetic and non-photosynthetic Eukaryotes
alike.
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Keywords
Botany, Biology--Cell, Plant Physiology
Citation
Uhrig, R. (2013). Characterization of Novel Shewanella- and Rhizobiales-Like PPP-Family Protein Phosphatases from Arabidopsis thaliana (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/25282