Assessment of the integral membrane protein topology in living cells

The bimolecular fluorescence complementation ( BiFC) phenomenon has been successfully applied for in vivo protein - protein interaction studies and protein tagging analysis. Here we report a novel BiFC- based technique for investigation of integral membrane protein topology in living plant cells. This technique relies on the formation of a fluorescent complex between a non- fluorescent fragment of the yellow fluorescent protein ( YFP) targeted into a specific cellular compartment and a counterpart fragment attached to the integral membrane protein N- or C- terminus or inserted into the internal loop( s). We employed this technique for topological studies of beet yellows virus- encoded p6 membrane- embedded movement protein, a protein with known topology, and the potato mop- top virus- encoded integral membrane TGBp2 protein with predicted topology. The results confirm that p6 is a type III integral transmembrane protein. Using a novel method, the central hydrophilic region of TGBp2 was localized into the ER lumen, whereas the N- and C- termini localized to the cytosol. We conclude that the BiFC- based reporter system for membrane protein topology analysis is a relatively fast and efficient method that can be used for high- throughput analysis of proteins integrated into the endoplasmic reticulum in living plant cells.