Moorhead, GregJohnson, Jayde Jarin2025-01-152025-01-152025-01-09Johnson, J. (2025). Biochemical characterization of the Arabidopsis chloroplast localized phosphatase Shewanella-like protein phosphatase 1 (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.https://hdl.handle.net/1880/120424Shewanella-like protein phosphatase 1 (SLP1) is a newly discovered member of the phosphoprotein phosphatase class of protein phosphatases. In Arabidopsis, SLP1 was previously confirmed to be chloroplast localized. Mass spectrometry based phosphoproteomics analysis of different Arabidopsis lines identified 126 high confidence SLP1 substrates, all of which were predicted/known chloroplast proteins. Identification of 126 substrates suggests SLP1 is a major regulator of chloroplast metabolism. Analysis of the substrates indicated SLP1 preferentially dephosphorylates serine or threonine near acidic residues. Similarly, a chloroplast localized protein kinase, casein kinase 2α4 (CK2), prefers acidic substrate motifs. Interestingly, many of CK2’s substrates are also SLP1 substrates, suggesting SLP1 and CK2 act antagonistically to balance chloroplast phosphostatus. CK2 has been studied for decades, with SLP1 studies beginning only recently. Herein I build on previous biochemical characterizations of SLP1 using phosphorylated peptides in enzyme assays. I use reagents to simulate diurnal fluctuations of redox, pH, and metabolite/protein concentrations in the stroma in vitro and found that SLP1 is activated by chloroplast thioredoxin m. I use structural prediction tools to connect changes in SLP1 activity to structural features such as a predicted disulfide bond. Predictions also suggest which residues may be responsible for preferential dephosphorylation at acidic motifs. The structural predictions guided SLP1 bioinformatics as novel regions of SLP1 were predicted and confirmed through sequence analysis of SLP1 homologues. Then, I explore the SLP1 substrate Calvin Cycle Protein of 12 kDa (CP12), as an example for how SLP1 phosphorylation may play a role in the regulating the Calvin Cycle. I reconstituted the inhibition complex of glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, with wild-type and mutant CP12s in vitro. Using size exclusion chromatography, I observed that the complex formed using phosphomimetic CP12T116D elutes differently from wild-type. Lastly, I generate a proximity labelling construct to explore SLP1 protein-protein interactions. I demonstrate that SLP1 substrates can be labelled with biotin when transiently expressed in tobacco plants. The work here builds on previous studies, identifying areas of chloroplast biology where redox regulation and reversible protein phosphorylation intersect.enUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.PhosphorylationChloroplastArabidopsisRedoxBiochemistrydoctoral thesis