PHOSPHORYLATION AND BEYOND: EXPLORATION OF TOR-MEDIATED PTM SIGNALING IN CHLAMYDOMONAS REINHARDTII

Target of rapamycin (TOR) is a highly conserved master regulatory kinase involved in the control of most essential biological processes including cell growth, nutrient sensing, and autophagy. While TOR is well-studied in mammalian species and yeast, comparatively little is known about its regulatory roles in other organisms, particularly in phototrophs. To fill this knowledge gap, the plant community is applying -omic strategies to assess the role of TOR in regulating metabolic pathways, particularly proteomics, which provides insight into expression levels and modifications that is missing in other techniques. Post-translational modifications (PTMs) to activate/deactivate functional proteins are an essential component of cellular signaling used by TOR and other regulators. This has propelled innovations in PTM analysis to probe metabolic pathways. Chlamydomonas reinhardtii is a model phototroph that is easily culturable and has a fully sequenced genome, making it an attractive organism for studying algal TOR signaling. The aim of this dissertation is to establish and apply PTM-focused proteomic methods in Chlamydomonas to characterize the role of protein phosphorylation and reversible oxidation in TOR’s regulation of signaling networks.First, this work investigates reversible oxidation sites under the control of TOR in Chlamydomonas through a quantitative inhibition study (Chapter 2). Next, a quantitative workflow for phosphopeptide analysis in Chlamydomonas that can be used to assess phosphorylative TOR signaling is described in detail with technical replicates to assess its overall reproducibility (Chapter 3). Application of the quantitative phosphoproteomic pipeline was then employed to study the impact of inositol polyphosphates on TOR signaling (Chapter 4). Techniques for performing in vitro kinase screening to identify direct targets of phosphorylation, are discussed including heterologous expression of kinase samples, preparation of a library of potential targets, screening parameters, and sample preparation for LC-MS/MS analysis (Chapter 5). This workflow provides insight into signaling pathways that cannot be gleaned from proteomic in vivo studies alone. Using these techniques, this kinase screening platform was validated using the pyruvate dehydrogenase kinase of Arabidopsis thaliana, and then applied to attempt to identify putative direct targets of Chlamydomonas TOR (Chapter 6). While this technique was unable to identify direct TOR targets, the success of the validation suggests that with further optimization of the expression and purification of the kinase construct, screening of TOR may be possible. Combined, the work shared herein highlights the importance of TOR in algal signaling and provides valuable information on the regulatory modifications under TOR control.Doctor of Philosoph