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Loose interaction between glyceraldehyde‐3‐phosphate dehydrogenase and phosphoglycerate kinase revealed by fluorescence resonance energy transfer–fluorescence lifetime imaging microscopy in living cells
Author(s) -
Tomokuni Yosuke,
Goryo Kenji,
Katsura Ayako,
Torii Satoru,
Yasumoto Kenichi,
Kemnitz Klaus,
Takada Mamiko,
Fukumura Hiroshi,
Sogawa Kazuhiro
Publication year - 2010
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2010.07561.x
Subject(s) - förster resonance energy transfer , glyceraldehyde 3 phosphate dehydrogenase , phosphoglycerate kinase , glyceraldehyde , dehydrogenase , cytoplasm , biophysics , fluorescence , biochemistry , chemistry , glycolysis , microbiology and biotechnology , biology , enzyme , physics , quantum mechanics
Loose interaction between the glycolytic enzymes glyceraldehyde‐3‐phosphate 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 (uniprotkb: P04406 ) by anti tag coimmunoprecipitation ( MI:0007 ) • MINT‐7386573 : GAPDH (uniprotkb: P04406 ) and PGK (uniprotkb: P00558 ) bind ( MI:0407 ) by fluorescent resonance energy transfer ( MI:0055 ) • MINT‐7386590 : GAPDH (uniprotkb: P04406 ) and GAPDH (uniprotkb: P04406 ) bind ( MI:0407 ) by fluorescent resonance energy transfer ( MI:0055 )