Elsevier, Biochemical and Biophysical Research Communications, 4(450), p. 1587-1592
DOI: 10.1016/j.bbrc.2014.07.046
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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.