Inorganic
polyphosphate (
polyP) is a linear
polymer of
orthophosphate and has many
biological functions in prokaryotic and eukaryotic organisms. To investigate
polyP localization, we developed a novel technique using the affinity of the recombinant
polyphosphate binding domain (PPBD) of
Escherichia coli exopolyphosphatase to
polyP. An
epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium binding assay of PPBD revealed its high affinity for long-chain
polyP and its weak affinity for short-chain
polyP and
nucleic acids. To directly demonstrate
polyP localization in Saccharomyces cerevisiae on resin sections prepared by rapid freezing and freeze-substitution, specimens were labeled with PPBD containing an
epitope tag and then the
epitope tag was detected by an indirect immunocytochemical method. A goat anti-mouse
immunoglobulin G antibody conjugated with Alexa 488 for
laser confocal microscopy or with
colloidal gold for transmission electron microscopy was used. When the S. cerevisiae was cultured in yeast extract-
peptone-
dextrose medium (10 mM
phosphate) for 10 h,
polyP was distributed in a dispersed fashion in vacuoles in successfully cryofixed cells. A few
polyP signals of the labeling were sometimes observed in cytosol around vacuoles with electron microscopy. Under our experimental conditions,
polyP granules were not observed. Therefore, it remains unclear whether the method can detect the granule form. The method directly demonstrated the localization of
polyP at the electron microscopic level for the first time and enabled the visualization of
polyP localization with much higher specificity and resolution than with other conventional methods.