title: Loose interaction between glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase revealed by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells.
abstract: 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.
authors.affiliation: Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Tomokuni
authors.forename: Kenji
authors.initials: K
authors.lastname: Goryo
authors.forename: Ayako
authors.initials: A
authors.lastname: Katsura
authors.forename: Satoru
authors.initials: S
authors.lastname: Torii
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Yasumoto
authors.forename: Klaus
authors.initials: K
authors.lastname: Kemnitz
authors.forename: Mamiko
authors.initials: M
authors.lastname: Takada
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fukumura
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Sogawa
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005455:Fluorescent Antibody Technique; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010735:Phosphoglycerate Kinase / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010735:Phosphoglycerate Kinase