Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors.
Precise and dynamic measurement of intracellular metabolite levels has been hampered by difficulties in differentiating between adsorbed and imported fractions and the subcellular distribution between cytosol, endomembrane compartments and mitochondria. In the present study, genetically encoded FRET (Förster resonance energy transfer)-based sensors were deployed for dynamic measurements of free cytosolic glucose and ATP with varying external supply and in glucose-transport mutants. Moreover, by using the FRET sensors in a microfluidic platform, we were able to monitor in vivo changes of intracellular free glucose in individual yeast cells. We demonstrate the suitability of the FRET sensors for gaining physiological insight by demonstrating that free intracellular glucose and ATP levels are reduced in a hxt5Δ hexose-transporter mutant compared with wild-type and other hxtΔ strains.
Bermejo
Clara
C
Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street., Stanford, CA 94305, USA.
Haerizadeh
Farzad
F
Takanaga
Hitomi
H
Chermak
Diane
D
Frommer
Wolf B
WB
D000255:Adenosine Triphosphate / Q000378:metabolism*; D015374:Biosensing Techniques; D003600:Cytosol / Q000378:metabolism*; D017931:DNA Primers; D004734:Energy Metabolism; D004784:Environmental Monitoring / Q000379:methods / Q000592:standards; D031541:Fluorescence Resonance Energy Transfer; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D018613:Microscopy, Confocal; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D017931:DNA Primers; C466910:HXT5 protein, S cerevisiae; D009004:Monosaccharide Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D005947:Glucose