Title: Direct labeling of polyphosphate at the ultrastructural level in Saccharomyces cerevisiae using affinity of the polyphosphate binding domain of Escherichia coli exopolyphosphatase

Inorganic polyphosphate (poly P) is a linear polymer of orthophosphate and has many biological functions in prokaryotic and eukaryotic organisms. To investigate poly P localization, we developed a novel technique using the affinity of the recombinant polyphosphate binding domain (PPBD) of Escherichia coli exopolyphosphatase to poly P. An epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium-binding assay of PPBD revealed its high affinity for long-chain poly P and its weak affinity for short-chain poly P and nucleic acids. To directly demonstrate poly P localization in Saccharomyces cerevisiae on resin sections prepared by rapid-freeze 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 IgG 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 YPD medium (10 mM phosphate) for 10 hours, poly P was distributed in a dispersed fashion in vacuoles in successfully cryofixed cells. A few poly P signals of the labeling were sometimes observed in cytosol around vacuoles with electron microscopy. Under our experimental conditions, poly P granules were not observed. Therefore, it remains unclear whether the method can detect the granule form. The method directly demonstrated the localization of poly P at the electron microscopic level for the first time and enabled the visualization of poly P localization with much higher specificity and resolution than with other conventional methods.