title: Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER.
abstract: The endoplasmic reticulum (ER) is an essential organelle for cellular metabolic homeostasis including folding and maturation of secretory and membrane proteins. Disruption of ER proteostasis has been implicated in the pathogenesis of various diseases such as diabetes and neurodegenerative diseases. The ER redox state, which is an oxidative environment suitable for disulfide-bond formation, is essential for ER protein quality control. Hence, detection of the ER redox state, especially in living cells, is essential to understand the mechanism by which the redox state of the ER is maintained. However, methods to detect the redox state of the ER have not been well-established because of inefficient folding and stability of roGFP variants with oxidative redox potential like roGFP-iL. Here we have improved the folding efficiency of ER-targeted roGFP-iL (ERroGFP-iL) in cells by introducing superfolder GFP (sfGFP) mutations. Four specific amino acid substitutions (S30R, Y39N, T105N and I171V) greatly improved folding efficiency in Escherichia coli and in the ER of HeLa cells, as well as the thermostability of the purified proteins. Introduction of these mutations also enhanced the dynamic range for redox change both in vitro and in the ER of living cells. ER-targeted roGFP-S4 (ERroGFP-S4) possessing these four mutations could detect physiological redox changes within the ER. ERroGFP-S4 is therefore a novel probe suitable for monitoring redox change in the ER. ERroGFP-S4 can be applied to detect aberrant ER redox states associated with various pathological conditions and to identify the mechanisms used to maintain the redox state of the ER.
authors.affiliation: Research Unit for Physiological Chemistry, the Center for the Promotion of Interdisciplinary Education and Research, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan houseki.jun.5v@kyoto-u.ac.jp.
authors.forename: Jun
authors.initials: J
authors.lastname: Hoseki
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Asami
authors.initials: A
authors.lastname: Oishi
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Fujimura
authors.affiliation: Research Unit for Physiological Chemistry, the Center for the Promotion of Interdisciplinary Education and Research, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Sakai
mesh_terms: D019943:Amino Acid Substitution; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins
keywords: endoplasmic reticulum (ER) redox state; glutathione; protein quality control; redox probe