A novel analytical method for in vivo phosphate tracking

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.