Live-cell topology assessment of URG7, MRP6(102) and SP-C using glycosylatable green fluorescent protein in mammalian cells
2014 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 450, no 4, 1587-1592 p.Article in journal (Refereed) Published
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(102), SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in C-in, form. MRP6(102) and SP-C(Leu/Val) are inserted into the membrane in C-out form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.
Place, publisher, year, edition, pages
2014. Vol. 450, no 4, 1587-1592 p.
Endoplasmic reticulum, Membrane protein topology, Protein orientation, GFP, N-linked glycosylation
IdentifiersURN: urn:nbn:se:su:diva-107994DOI: 10.1016/j.bbrc.2014.07.046ISI: 000341338100058OAI: oai:DiVA.org:su-107994DiVA: diva2:752947