Thiol-based regulation of oxidative protein folding in the ER of plants

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Secreted proteins are essential for a cell to interact with its environment. The folding of most secretory proteins in the endoplasmic reticulum (ER) strictly requires the catalyzed formation of intramolecular disulfide bonds between cysteines. The disulfides are critical for protein structure and function, which makes their formation indispensable for survival. Protein disulfide isomerase (PDI) and ER thiol oxidases (ERO) together make up the oxidative protein folding machinery of the ER. They constitute a disulfide relay system for the transfer of electrons from cysteines of nascent protein substrates to molecular oxygen. This machinery needs to be dynamically regulated, as the demand for oxidative protein folding in the ER can vary dramatically, depending on developmental stage and environmental conditions. As sessile organisms plants are frequently exposed to particularly severe environmental changes, which necessitate rapid acclimation responses on cellular level. In yeast and mammals, the activity of the folding machinery is modulated by regulatory thiol switches on the EROs, allowing rapid posttranslational control. While the control of oxidative protein folding is not understood in plants, the ERO isoforms of plants contain several additional cysteines as compared to their yeast and mammalian counterparts. Based on their position in the ERO protein these cysteines are likely to constitute an additional, plant-specific level of ERO redox control by stepwise activation of multiple thiol switches or the formation of alternative disulfides. To address the thiol-based regulation of oxidative protein folding in the ER of plants Arabidopsis thaliana and Physcomitrella patens will be employed as model systems. A combination of knockout and knockdown for the two ERO isoforms in Arabidopsis results in a severe sensitivity towards DTT and under non-stress conditions a pronounced insensitivity towards ethylene which identifies the ethylene receptor ETR1 as a sensitive target of ERO activity. The goal of this project is to identify and to characterize regulatory thiol switches in plant ERO proteins and their interaction with PDIs as switch operators as well as PDI target proteins. Biochemical analysis of ERO regulation will be combined with in vivo studies of the thiol redox poise in the ER. As dynamic measurements of the glutathione redox potential and H2O2 in the ER are still limited by a lack of specific and sensitive sensors, improved fluorescent protein-based sensor variants will be developed for oxidizing redox environments. These attempts will build on improved mechanistic understanding of roGFP-interactions and modelling of protein-protein interactions. Being able to image redox state and H2O2 levels in the ER will provide a major step forward for quantitative redox analysis and generate novel depth of insight into the role of ERO and its regulatory thiols in setting ER redox homeostasis.