title: Real-time monitoring of redox changes in the mammalian endoplasmic reticulum.
abstract: Redox-sensitive GFPs with engineered disulphide bonds have been used previously to monitor redox status in the cytosol and mitochondria of living cells. The usefulness of these redox probes depends on the reduction potential of the disulphide, with low values suiting the cytosol and mitochondrion, and higher values suiting the more oxidising environment of the endoplasmic reticulum (ER). Here, we targeted a modified redox-sensitive GFP (roGFP1-iL), with a relatively high reduction potential, to the ER of mammalian cells. We showed that the disulphide is partially oxidised, allowing roGFP1-iL to monitor changes in ER redox status. When cells were treated with puromycin, the redox balance became more reducing, suggesting that the release of nascent chains from ribosomes alters the ER redox balance. In addition, downregulating Ero1α prevented normal rapid recovery from dithiothreitol (DTT), whereas downregulating peroxiredoxin IV had no such effect. This result illustrates the contribution of the Ero1α oxidative pathway to ER redox balance. This first report of the use of roGFP to study the ER of mammalian cells demonstrates that roGFP1-iL can be used to monitor real-time changes to the redox status in individual living cells.
authors.affiliation: College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Marcel
authors.initials: M
authors.lastname: van Lith
authors.forename: Shweta
authors.initials: S
authors.lastname: Tiwari
authors.forename: John
authors.initials: J
authors.lastname: Pediani
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Bulleid
mesh_terms: D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D004220:Disulfides; D008562:Membrane Glycoproteins; D049452:Green Fluorescent Proteins; C405327:ERO1A protein, human; D010088:Oxidoreductases