Michelle Schlösser (Bonn University)
A: Different algorithms predict different topologies for AtGRXC4 or even secretion as a soluble protein. B: Schematic illustration of redox-based topology analysis (ReTA) in which GRXC3 and GRXC4 were fused to roGFP2 at their N- and C-termini. Binary readout of roGFP2 fluorescence directly indicates localization in the ER (oxidized) or cytosol (reduced). C: Expression of N- and C-terminal fusions of GRXC4 with roGFP2 in Nicotiana tabacum. After acquisition of two images with excitation at 405 nm (magenta) and 488 nm (green), the colour of the superimposed single images (merge) indicates the orientation of the protein in the membrane (green: cytosol; magenta: ER lumen). Images: Michelle Schlößer
Michelle Schlösser's Master thesis was awarded with the Prize for the Best Plant Science Master Thesis, which was carried out at Bonn University in the year 2021
Title: Characterization of the ER-localized glutaredoxins GRXC3 and GRXC4 in Arabidopsis
Arabidopsis thaliana GRXC3 and GRXC4 are type II membrane proteins in the ER lumen with high in vitro oxidative activity towards roGFP2 as a substrate, which suggests that the two oxidoreductases are involved in oxidation of protein thiols in the ER.
Class I glutaredoxins (GRXs) are small oxidoreductases that use reduced glutathione (GSH) as a co-factor to reduce or oxidize substrate proteins. The Arabidopsis genome encodes six class I GRXs of which GRXC3 and GRXC4 are generally assumed to be secreted. Some algorithms for prediction of transmembrane proteins, however, predict both proteins as single-spanning membrane proteins with an N-terminal transmembrane domain (TMD) albeit with different orientations in the membrane as either type I or type II proteins. While such a membrane anchor could prevent the proteins from being secreted further functional analysis then requires also knowledge about the protein orientation relative to the respective membrane. Fusion of GRXC3 and GRXC4 with redox-sensitive GFP2 (roGFP2) at their N- and C-termini and expression in tobacco indicates both proteins as type II ER membrane proteins with their catalytic domains oriented towards the lumen. Additional studies with only the TMDs fused to roGFP2 showed that the TMDs alone are sufficient to keep roGFP2 in the ER and thus are likely to act as membrane anchors that simultaneously restrict the localization of both proteins to the ER.
To further study the function of GRXC3 and GRXC4, both proteins were synthesized as recombinant proteins and purified. Like their cytosolic siblings, both proteins are capable of reversibly reducing and oxidizing roGFP2 as an artificial substrate protein. Surprisingly, however, in comparison to cytosolic GRXC1 the catalytic properties of GRXC3 and GRXC4 appear to be shifted towards the oxidizing reactions in which addition of glutathione disulfide leads to oxidation of roGFP2.
Taken together, these studies show a revised localization of GRXC3 and GRXC4 with the catalytic functions being likely involved in oxidative protein folding in the ER. The results of this Master thesis thus set the base for further functional analysis of these proteins.
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Michelle Schlösser conducted this work at the Institute of Crop Science and Resource Conservation (INRES) under supervision of Dr. José Ugalde and Prof. Dr. Andreas Meyer.