skip to main content

SciTech ConnectSciTech Connect

Title: Live-cell topology assessment of URG7, MRP6{sub 102} and SP-C using glycosylatable green fluorescent protein in mammalian cells

Highlights: • Glycosylatable GFP (gGFP) is developed for the use in mammalian cells. • gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. • Differential fluorescence/glycosylation pattern probes membrane protein topology. • Membrane topology of URG7, MRP6{sub 102}, and SP-C was determined by gGFP tagging in vivo. - 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, MRP6{sub 102}, SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in C{sub in} form. MRP6{sub 102} and SP-C(Leu/Val) are inserted into the membrane in C{sub out} form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [4] ;  [5] ;  [6] ;  [2] ;  [1]
  1. School of Biological Sciences, Seoul National University, Seoul 151-747 (Korea, Republic of)
  2. Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm (Sweden)
  3. Department of Sciences, University of Basilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza (Italy)
  4. Karolinska Institutet, Dept of Neurobiology, Care Sciences and Society, Novum 5th Floor, 141 86 Stockholm (Sweden)
  5. (Sweden)
  6. (Estonia)
Publication Date:
OSTI Identifier:
22416699
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 450; Journal Issue: 4; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANIMAL CELLS; CELL MEMBRANES; DMSO; ENDOPLASMIC RETICULUM; FLUORESCENCE; GENES; HEPATITIS; HEPATOMAS; IN VIVO; LUNGS; MEMBRANE PROTEINS; PROBES; TOPOLOGY; VIRUSES