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.
Zamyatnin
Andrey A
AA
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
Solovyev
Andrey G
AG
Bozhkov
Peter V
PV
Valkonen
Jari P T
JP
Morozov
Sergey Yu
SY
Savenkov
Eugene I
EI
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