Live-cell topology assessment of URG7, MRP6{sub 102} and SP-C using glycosylatable green fluorescent protein in mammalian cells
- School of Biological Sciences, Seoul National University, Seoul 151-747 (Korea, Republic of)
- Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm (Sweden)
- Department of Sciences, University of Basilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza (Italy)
- Karolinska Institutet, Dept of Neurobiology, Care Sciences and Society, Novum 5th Floor, 141 86 Stockholm (Sweden)
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.
- OSTI ID:
- 22416699
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 450, 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); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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