title: Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 1.
abstract: Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress.
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, SE-17177 Stockholm, Sweden.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Shimoji
authors.affiliation: Department of Neurochemistry, Stockholm University, SE-10691 Stockholm, Sweden.
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Figueroa
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, SE-17177 Stockholm, Sweden.
authors.forename: Etienne
authors.initials: E
authors.lastname: Neve
authors.affiliation: Department of Neurochemistry, Stockholm University, SE-10691 Stockholm, Sweden.
authors.forename: Danuta
authors.initials: D
authors.lastname: Maksel
authors.affiliation: Department of Neurochemistry, Stockholm University, SE-10691 Stockholm, Sweden.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Imreh
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, SE-17177 Stockholm, Sweden. Electronic address: ralf.morgenstern@ki.se.
authors.forename: Ralf
authors.initials: R
authors.lastname: Morgenstern
authors.affiliation: Department of Neurochemistry, Stockholm University, SE-10691 Stockholm, Sweden.
authors.forename: Einar
authors.initials: E
authors.lastname: Hallberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology
chemical_list: C122107:microsomal glutathione S-transferase-I; D005982:Glutathione Transferase
keywords: Dual distribution; Glutathione transferase; Subcellular targeting