Assessment of the integral membrane protein topology in living cells

Authors

  • Andrey A. Zamyatnin Jr,

    Corresponding author
    1. Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), Genetic Centre, Box 7080, SE-750 07 Uppsala, Sweden,
      *(fax +46 18 673279; e-mail andrey.zamyatnin@vbsg.slu.se).
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  • Andrey G. Solovyev,

    1. A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia, and
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  • Peter V. Bozhkov,

    1. Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), Genetic Centre, Box 7080, SE-750 07 Uppsala, Sweden,
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  • Jari P.T. Valkonen,

    1. Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), Genetic Centre, Box 7080, SE-750 07 Uppsala, Sweden,
    2. Department of Applied Biology, PO Box 27, FIN-00014, University of Helsinki, Finland
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  • Sergey Yu. Morozov,

    1. A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia, and
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  • Eugene I. Savenkov

    1. Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), Genetic Centre, Box 7080, SE-750 07 Uppsala, Sweden,
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*(fax +46 18 673279; e-mail andrey.zamyatnin@vbsg.slu.se).

Summary

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.

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