title: Ternary complex formation of pVHL, elongin B and elongin C visualized in living cells by a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique.
abstract: The tumor suppressor von Hippel-Lindau (VHL) gene product forms a complex with elongin B and elongin C, and acts as a recognition subunit of a ubiquitin E3 ligase. Interactions between components in the complex were investigated in living cells by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM). Elongin B-cerulean or cerulean-elongin B was coexpressed with elongin C-citrine or citrine-elongin C in CHO-K1 cells. FRET signals were examined by measuring a change in the fluorescence lifetime of donors and by monitoring a corresponding fluorescence rise of acceptors. Clear FRET signals between elongin B and elongin C were observed in all combinations, except for the combination of elongin B-cerulean and citrine-elongin C. Although similar experiments to examine interaction between pVHL30 and elongin C linked to cerulean or citrine were performed, FRET signals were rarely observed among all the combinations. However, the signal was greatly increased by coexpression of elongin B. These results, together with results of coimmunoprecipitation experiment using pVHL, elongin C and elongin B, suggest that a conformational change of elongin C and/or pVHL was induced by binding of elongin B. The conformational change of elongin C was investigated by measuring changes in the intramolecular FRET signal of elongin C linked to cerulean and citrine at its N- and C-terminus, respectively. A strong FRET signal was observed in the absence of elongin B, and this signal was modestly increased by coexpression of elongin B, demonstrating that a conformation change of elongin C was induced by the binding of elongin B.
authors.affiliation: Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Aoba-Ku Sendai, Japan.
authors.forename: Koshi
authors.initials: K
authors.lastname: Kinoshita
authors.forename: Kenji
authors.initials: K
authors.lastname: Goryo
authors.forename: Mamiko
authors.initials: M
authors.lastname: Takada
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Tomokuni
authors.forename: Teijiro
authors.initials: T
authors.lastname: Aso
authors.forename: Heiwa
authors.initials: H
authors.lastname: Okuda
authors.forename: Taro
authors.initials: T
authors.lastname: Shuin
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fukumura
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Sogawa
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000075065:Elongin; D031541:Fluorescence Resonance Energy Transfer; D008856:Microscopy, Fluorescence; D011487:Protein Conformation; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000075065:Elongin; D014157:Transcription Factors; D051794:Von Hippel-Lindau Tumor Suppressor Protein