title: Live-cell topology assessment of URG7, MRP6₁₀₂ and SP-C using glycosylatable green fluorescent protein in mammalian cells.
abstract: Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6102, SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in Cin form. MRP6102 and SP-C(Leu/Val) are inserted into the membrane in Cout form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.
authors.affiliation: School of Biological Sciences, Seoul National University, Seoul 151-747, South Korea.
authors.forename: Hunsang
authors.initials: H
authors.lastname: Lee
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Patricia
authors.initials: P
authors.lastname: Lara
authors.affiliation: Department of Sciences, University of Basilicata, Viale dell'Ateneo Lucano 10, 85100 Potenza, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Ostuni
authors.affiliation: Karolinska Institutet, Dept of Neurobiology, Care Sciences and Society, Novum 5th Floor, 141 86 Stockholm, Sweden.
authors.forename: Jenny
authors.initials: J
authors.lastname: Presto
authors.affiliation: Karolinska Institutet, Dept of Neurobiology, Care Sciences and Society, Novum 5th Floor, 141 86 Stockholm, Sweden; Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, 751 23 Uppsala, Sweden; Institute of Mathematics and Natural Sciences, Tallinn University, Narva mnt 25, 101 20 Tallinn, Estonia.
authors.forename: Janne
authors.initials: J
authors.lastname: Johansson
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: IngMarie
authors.initials: I
authors.lastname: Nilsson
authors.affiliation: School of Biological Sciences, Seoul National University, Seoul 151-747, South Korea. Electronic address: joy@snu.ac.kr.
authors.forename: Hyun
authors.initials: H
authors.lastname: Kim
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence
chemical_list: D049452:Green Fluorescent Proteins
keywords: Endoplasmic reticulum; GFP; Membrane protein topology; N-linked glycosylation; Protein orientation