Dissecting the function of Arabidopsis chloroplast, mitochondrial and cytosolic bacterial-like protein phosphatases

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Proteins are the building blocks and molecular machines of cells. Synthesizing a protein is energy and resource consuming, so it is no surprise that cells have evolved ways to precisely regulate the function of proteins. A protein's job or role in the cell can be turned on or off by adding specific chemical groups to specific amino acids of the protein, essentially making a molecular switch to turn function on and off. Although many different chemical groups can be specifically added to proteins, the most common form of a molecular switch is the addition or removal of phosphoryl group to a serine, threonine or tyrosine residue. This addition is covalent and added by enzymes known as protein kinases, and removed by protein phosphatases. My laboratory has studied the latter group of enzymes for two decades applying biochemistry, molecular biology, bioinformatics and cell biology to understand their precise roles in cells. Most recently we have begun to characterize a group of bacterial-like' phosphatases and consistent with their origin, some of them reside in the mitochondrion and chloroplast. By employing knockout lines in Arabidopsis, we were able to perform quantitative phospho-proteomics and demonstrate that this method is a powerful tool to uncover substrates of protein phosphatases. This proved enormously successful and we have determined that the tyrosine specific phosphatase RLPH2 specifically targets the phospho-tyrosine in the activation loop (a TxY site) of the cytosolic D-group MAP kinases (MAPKs). In addition, we have identified many substrates of the chloroplast phosphatase SLP1. We will now use the same approach to uncover substrates of the related mitochondrial inter-membrane space phosphatase SLP2. In addition we have explored the phospho-status of the chloroplast proteome (proteins) and determined that SLP1 operates primarily in the dark. How it is regulated between light and dark will be further explored in this proposal.***