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A novel analytical method for in vivo phosphate tracking.

Hong Gu·Sylvie Lalonde·Sakiko Okumoto·Loren L Looger·Anne Marie Scharff-Poulsen·Arthur R Grossman·Jens Kossmann·Iver Jakobsen·Wolf B Frommer
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Abstract

Genetically-encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (P(i)) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate-binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows P(i)-dependent increases in FRET efficiency. FLIPPi affinity mutants cover P(i) changes over eight orders of magnitude. COS-7 cells co-expressing a low-affinity FLIPPi and a Na(+)/P(i) co-transporter exhibited FRET changes when perfused with 100 microM P(i), demonstrating concentrative P(i) uptake by PiT2. FLIPPi sensors are suitable for real-time monitoring of P(i) metabolism in living cells, providing a new tool for fluxomics, analysis of pathophysiology or changes of P(i) during cell migration.

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AnimalsBacterial ProteinsBase SequenceBinding SitesBiosensing TechniquesCHO CellsCOS CellsChlorocebus aethiopsCricetinaeDNA, BacterialFluorescence Resonance Energy TransferFluorescent DyesGreen Fluorescent ProteinsKineticsLuminescent ProteinsModels, MolecularMutagenesis, Site-DirectedPhosphate-Binding ProteinsPhosphatesProtein ConformationProtein EngineeringRecombinant Fusion ProteinsSequence DeletionSynechococcusJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.

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Hong Gu, Sylvie Lalonde, Sakiko Okumoto, Loren L Looger, Anne Marie Scharff-Poulsen, Arthur R Grossman, Jens Kossmann, Iver Jakobsen, Wolf B Frommer. 2006-10-02. A novel analytical method for in vivo phosphate tracking.. https://doi.org/10.1016/j.febslet.2006.09.048

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