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

Koshi Kinoshita, Kenji Goryo, Mamiko Takada, Yosuke Tomokuni, Teijiro Aso, Heiwa Okuda, Taro Shuin, Hiroshi Fukumura, Kazuhiro Sogawa

Research output: Contribution to journalArticle

12 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)5567-5575
Number of pages9
JournalFEBS Journal
Volume274
Issue number21
DOIs
Publication statusPublished - 2007 Nov 1

Keywords

  • Conformation change
  • FRET-FLIM
  • Live cell imaging
  • Protein complex
  • Ubiquitin ligase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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