A Fast Metabolic Sensor for in Vivo Cytosolic Phosphate Concentration in Saccharomyces Cerevisiae

Eukaryotic metabolism consists of a complex network of enzymatic reactions and transport processes which are distributed over different subcellular compartments. Currently, available measurement techniques allow to measure whole cell amounts which in several cases is not sufficient to describe the in-vivo kinetics in different compartments, which are driven by compartment specific concentrations. Phosphate (Pi) is considered an essential component for the metabolic behaviour of glycolysis. Specially, the cytosolic Pi level is important for the balance between the upper and lower glycolysis. One prominent example supporting the role of Pi is the disregulation of glycolysis in the tps1 knockout strain of Saccharomyces cerevisiae. TPS1 is expected to act as cytosolic phosphate recovery mechanism, building a safe-guard to glycolysis. Understanding the concentration of Pi in different compartments under dynamic conditions is critical to study the Pi metabolism, function and regulation. 

Fractionation, permeabilization, in vivo NMR, and metabolic sensor reactions have been applied to monitor the ratios of conserved moieties in subcellular compartments. Here we developed a method that enables to monitor the dynamic cytosolic Pi in S. cerevisiae by an equilibrium sensor reaction: maltose + Pi <=> D-glucose +D-glucose 1-phosphate. The required enzyme, maltose phosphorylase from L. sanfranciscensis was overexpressed in S. cerevisiae. Additionally, β-phosphoglucomutase from L. lactis was overexpressed to convert G1P to G6P which will have a higher concentration and is therefore measured more accurately. Furthermore, maltose hydrolase was knocked out to prevent a cycle. The cytosolic free Pi concentration can then be calculated from the measured intracellular glucose, G6P and maltose concentrations. 

The sensor was applied to study Pi level in S. cerevisiae in several different conditions. The cytosolic free Pi concentration in batch condition was 13.7mM in S. cerevisiae. We also studied the Pi under different Pi supply conditions: Pi-excess (glucose-limited) chemostat and Pi-limited chemostat at D=0.1h^-1 and under dynamic pulse conditions e.g. from glucose-limited to glucose excess and from Pi-limited to Pi-excess condition. 

There was a significant difference between the cytosolic and whole cell concentration. The steady-state cytosolic free Pi concentration in glucose-limited condition was 14.7 mM, which corresponds to 31.4% of the whole cell free Pi concentration (46.8 mM). While in Pi-limited condition, the steady-state cytosolic free Pi concentration was 2.6 mM, which was about 15.7% of the whole cell Pi concentration (16.6 mM). Additionally, dynamic pulse experiments from glucose-limited to glucose excess and from Pi-limited to Pi-excess condition revealed fast dynamics of Pi. The cytosolic free Pi concentration increased 2.5 fold within 10 seconds and then decreased, which was unexpected as especially sugar-phosphate are rapidly formed in the first seconds. The Pi balance was constructed from cytosolic Pi, Pi in nucleotides, Pi in glycolytic intermediates, Pi in other measured intracellular metabolites, Pi in the vacuole which was calculated by using whole cell Pi minus cytosolic Pi, and polyPi. The results showed that the immediately increase of cytosolic Pi level was not caused by polyphosphate hydrolysis but due to the transport of Pi from the vacuole, as well as Pi uptake from the extracellular space. From these compartment specific Pi data hypothesis about the metabolic regulations can be derived and integrated into thermodynamic and kinetic models.