title: Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.
abstract: Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool.
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
authors.forename: Maria
authors.initials: M
authors.lastname: Tsachaki
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
authors.forename: Julia
authors.initials: J
authors.lastname: Birk
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Egert
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland. Electronic address: alex.odermatt@unibas.ch.
authors.forename: Alex
authors.initials: A
authors.lastname: Odermatt
mesh_terms: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000737:chemistry / Q000378:metabolism; D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000378:metabolism; D002463:Cell Membrane Permeability; D002470:Cell Survival; D003198:Computer Simulation; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015067:17-Hydroxysteroid Dehydrogenases; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase
keywords: 17β-hydroxysteroid dehydrogenase; Endoplasmic reticulum; Green-fluorescent protein; Live-imaging; Redox sensor; Topology