Loose interaction between glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase revealed by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells

Yosuke Tomokuni; Kenji Goryo; Ayako Katsura; Satoru Torii; Ken Ichi Yasumoto; Klaus Kemnitz; Mamiko Takada; Hiroshi Fukumura; Kazuhiro Sogawa

doi:10.1111/j.1742-4658.2010.07561.x

Loose interaction between glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase revealed by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells

Yosuke Tomokuni, Kenji Goryo, Ayako Katsura, Satoru Torii, Ken Ichi Yasumoto, Klaus Kemnitz, Mamiko Takada, Hiroshi Fukumura, Kazuhiro Sogawa

LIF - Molecular and Chemical Life Science

Research output: Contribution to journal › Article › peer-review

11 Citations (Scopus)

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.

Original language	English
Pages (from-to)	1310-1318
Number of pages	9
Journal	FEBS Journal
Volume	277
Issue number	5
DOIs	https://doi.org/10.1111/j.1742-4658.2010.07561.x
Publication status	Published - 2010 Mar

Keywords

FLIM
FRET
GAPDH
Loose interaction
PGK

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10.1111/j.1742-4658.2010.07561.x

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Tomokuni, Y., Goryo, K., Katsura, A., Torii, S., Yasumoto, K. I., Kemnitz, K., Takada, M., Fukumura, H., & Sogawa, K. (2010). Loose interaction between glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase revealed by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells. FEBS Journal, 277(5), 1310-1318. https://doi.org/10.1111/j.1742-4658.2010.07561.x

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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.",

keywords = "FLIM, FRET, GAPDH, Loose interaction, PGK",

author = "Yosuke Tomokuni and Kenji Goryo and Ayako Katsura and Satoru Torii and Yasumoto, {Ken Ichi} and Klaus Kemnitz and Mamiko Takada and Hiroshi Fukumura and Kazuhiro Sogawa",

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Tomokuni, Y, Goryo, K, Katsura, A, Torii, S, Yasumoto, KI, Kemnitz, K, Takada, M, Fukumura, H & Sogawa, K 2010, 'Loose interaction between glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase revealed by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells', FEBS Journal, vol. 277, no. 5, pp. 1310-1318. https://doi.org/10.1111/j.1742-4658.2010.07561.x

Loose interaction between glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase revealed by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells. / Tomokuni, Yosuke; Goryo, Kenji; Katsura, Ayako et al.
In: FEBS Journal, Vol. 277, No. 5, 03.2010, p. 1310-1318.

Research output: Contribution to journal › Article › peer-review

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T1 - Loose interaction between glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase revealed by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells

AU - Tomokuni, Yosuke

AU - Goryo, Kenji

AU - Katsura, Ayako

AU - Torii, Satoru

AU - Yasumoto, Ken Ichi

AU - Kemnitz, Klaus

AU - Takada, Mamiko

AU - Fukumura, Hiroshi

AU - Sogawa, Kazuhiro

PY - 2010/3

Y1 - 2010/3

N2 - 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.

AB - 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.

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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

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Keywords

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