Live-cell topology assessment of URG7, MRP6₁₀₂ and SP-C using glycosylatable green fluorescent protein in mammalian cells.

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 membranetopology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylationin the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas nofluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By usingmammalian 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 Cinform.MRP6102and SP-C(Leu/Val) are inserted into the membrane in Coutform. A minor population of untarget-ed SP-C is removed by proteasome dependent quality control system