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-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.
Tomokuni
Yosuke
Y
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Japan.
Goryo
Kenji
K
Katsura
Ayako
A
Torii
Satoru
S
Yasumoto
Ken-ichi
K
Kemnitz
Klaus
K
Takada
Mamiko
M
Fukumura
Hiroshi
H
Sogawa
Kazuhiro
K
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*
D018076:DNA, Complementary; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010735:Phosphoglycerate Kinase