GFP-nanobody-mCherry as a tool for identifying the topology of GFP-fused membrane proteins in intracellular organelles in cells

s: A key characteristic of membrane proteins in various intracellular organelles is their topology. The topology of green fluorescent protein (GFP)-tagged transmembrane proteins in membranous organelles can be elucidated using various methods, including protease protection and fluorescence protease protection assays. However, there is still a lack of a simple method to identify topology without disrupting the membrane of the plasma membrane and intracellular organelles, including endoplasmic reticulum and mitochondria. In this study, we demonstrate that GFP nanobody (GNb) fused to mCherry (GNb–mCherry) can be used to identify the topology of GFP-fused membrane proteins in cells. We first demonstrate that cytosolically expressed GNb– mCherry can discriminate cytoplasmic or extracellular or luminal side localization of GFP in GFP-fused plasma membrane, endoplasmic reticulum, Trans-Golgi network, lysosome or mitochondria-targeting proteins. Next, using this assay, we could determine the topology of PRMT8(N20)-GFP in the plasma membrane and mitochondria. Overall, GNb–mCherry can be a useful tool for identifying the topology of GFP-fused membrane proteins in intracellular organelles in cells.