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.
Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), Genetic Centre, Box 7080, SE-750 07 Uppsala, Sweden. andrey.zamyatnin@vbsg.slu.se
Andrey A
AA
Zamyatnin
Andrey G
AG
Solovyev
Peter V
PV
Bozhkov
Jari P T
JP
Valkonen
Sergey Yu
SY
Morozov
Eugene I
EI
Savenkov
D004721:Endoplasmic Reticulum / Q000737:chemistry; D007425:Intracellular Membranes / Q000737:chemistry; D008164:Luminescent Proteins / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D018515:Plant Leaves / Q000166:cytology / Q000648:ultrastructure; D010944:Plants / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D014026:Nicotiana / Q000166:cytology / Q000648:ultrastructure
D008164:Luminescent Proteins; D008565:Membrane Proteins