tailieunhanh - Báo cáo khoa học: Loose interaction between glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase revealed by fluorescence resonance energy transfer–fluorescence lifetime imaging microscopy in living cells

Loose interaction between the glycolytic enzymes glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) was visualized in living CHO-K1 cells by fluorescence resonance energy transfer (FRET), using time-domain fluorescence lifetime imaging microscopy. | Loose interaction between glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase revealed by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells Yosuke Tomokuni1 Kenji Goryo1 Ayako Katsura1 Satoru Torii1 Ken-ichi Yasumoto1 Klaus Kemnitz2 Mamiko Takada3 Hiroshi Fukumura3 and Kazuhiro Sogawa1 1 Department of Biomolecular Sciences Graduate Schoolof Life Sciences Tohoku University Aoba-ku Sendai Japan 2 EuroPhoton GmbH Berlin Germany 3 Department of Chemistry Graduate Schoolof Science Tohoku University Aoba-ku Sendai Japan Keywords FLIM FRET GAPDH loose interaction PGK Correspondence K. Sogawa Department of Biomolecular Sciences Graduate School of Life Sciences Tohoku University Aoba-ku Sendai 980-8578 Japan Fax 81 22 795 6594 Tel 81 22 795 6590 E-mail sogawa@ Received 14 April2009 revised 7 December 2009 accepted 24 December 2009 doi Loose interaction between the glycolytic enzymes glyceraldehyde-3-phos-phate dehydrogenase GAPDH and phosphoglycerate kinase PGK was visualized in living CHO-K1 cells by fluorescence resonance energy transfer FRET using time-domain fluorescence lifetime imaging microscopy. FRET between active tetrameric subunits of GAPDH linked to cerulean or citrine was observed and this FRET signal was significantly attenuated by coexpression of PGK. Also direct interaction between GAPDH-citrine and PGK-cerulean was observed by FRET. The strength of FRET signals between them was dependent on linkers that connect GAPDH to citrine and PGK to cerulean. A coimmunoprecipitation assay using hemagglutinin-tagged GAPDH and FLAG-tagged PGK coexpressed in CHO-K1 cells supported the FRET observation. Taken together these results demonstrate that a complex of GAPDH and PGK is formed in the cytoplasm of living cells. Structured digital abstract MINT-7386555 PGK uniprotkb P00558 physically interacts MI 0915 with GAPDH uni-protkb P04406 by anti tag .