Journal
FEBS LETTERS
Volume 580, Issue 25, Pages 5885-5893Publisher
WILEY
DOI: 10.1016/j.febslet.2006.09.048
Keywords
fluorescence energy transfer; phosphate starvation; biosensor; Synechococcus
Funding
- NIDDK NIH HHS [R01 DK079109-01, R01 DK079109] Funding Source: Medline
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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 mu M 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. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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