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

蛋白质是细胞的基石和分子机器。合成蛋白质需要消耗能量和资源，所以细胞进化出精确调节蛋白质功能的方法也就不足为奇了。通过在蛋白质的特定氨基酸中添加特定的化学基团，可以打开或关闭蛋白质在细胞中的工作或角色，本质上是制造一个分子开关来打开或关闭功能。虽然许多不同的化学基团可以特别地添加到蛋白质中，但分子开关最常见的形式是在丝氨酸、苏氨酸或酪氨酸残基上添加或去除磷酸化基团。这种添加物是共价的，由称为蛋白激酶的酶添加，并由蛋白磷酸酶去除。我的实验室对后一类酶进行了二十年的研究，应用生物化学、分子生物学、生物信息学和细胞生物学来了解它们在细胞中的确切作用。最近，我们已经开始描述一组类似细菌的磷酸酶，与它们的起源一致，其中一些存在于线粒体和叶绿体中。通过在拟南芥中使用敲除系，我们能够进行定量磷酸化蛋白质组学，并证明这种方法是发现蛋白磷酸酶底物的有力工具。这被证明是非常成功的，我们已经确定酪氨酸特异性磷酸酶RLPH2特异性靶向胞浆d组MAP激酶（MAPKs）激活环（一个TxY位点）中的磷酸酪氨酸。此外，我们已经确定了叶绿体磷酸酶SLP1的许多底物。我们现在将使用相同的方法来揭示相关线粒体膜间空间磷酸酶SLP2的底物。此外，我们还探索了叶绿体蛋白质组（蛋白质）的磷酸化状态，并确定SLP1主要在黑暗中运作。如何在光明和黑暗之间进行调节将在本提案中进一步探讨。

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.