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 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.
Gu
Hong
H
Carnegie Institution, Department of Plant Biology, 260 Panama Street, Stanford, CA 94305, USA.
Lalonde
Sylvie
S
Okumoto
Sakiko
S
Looger
Loren L
LL
Scharff-Poulsen
Anne Marie
AM
Grossman
Arthur R
AR
Kossmann
Jens
J
Jakobsen
Iver
I
Frommer
Wolf B
WB
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004269:DNA, Bacterial / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D028044:Phosphate-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D046940:Synechococcus / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins