Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-xL in living cells

Shuya Kasai, Shinji Kajimoto, Yuma Ito, Tomo Saito, Ken Ichi Yasumoto, Makio Tokunaga, Kumiko Sakata-Sogawa, Hiroshi Fukumura, Kazuhiro Sogawa

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color singlemolecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1a or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.

Original languageEnglish
Pages (from-to)291-296
Number of pages6
JournalJournal of biochemistry
Volume161
Issue number3
DOIs
Publication statusPublished - 2017 Jan 1

Keywords

  • Conformational change
  • Dual-color single molecule imaging
  • FLIM-FRET analysis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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