Direct Labeling of Polyphosphate at the Ultrastructural Level inSaccharomyces cerevisiaeby Using the Affinity of the Polyphosphate Binding Domain ofEscherichia coliExopolyphosphatase

Abstract

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.Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate (P i ) connected by high-energy bonds. PolyP occurs in a wide range of organisms, including prokaryotes and eukaryotes. PolyP has various biological functions; for example, it acts as a P i reservoir, as an alternative source of highenergy bonds, and as a buffer against alkaline conditions and metals (25). Furthermore, polyP also has regulatory functions such as competence for transformation (18), motility (36, 37), gene expression under stressed conditions (24,35,46), and protein degradation in amino acid starvation (28, 29) in prokaryotic organisms. The regulatory functions of polyP in eukaryotes are less clear, but some important facts are known. Recently, involvement of polyP in apoptosis (43) and enhancement of the mitogenic activities of acidic and basic fibroblast growth factors by polyP (45) have been suggested to occur in mammalian cells.The presence of polyP in cells can be visualized by staining with toluidine blue O (TBO) or 4Ј,6-diamidino-2-phenylindole (DAPI). DAPI is usually used for DNA detection, because blue fluorescence is apparent when the stained tissues are viewed under UV light. However, DAPI-polyP fluoresces yellow at high concentrations when viewed under UV (48). These staining methods have often been used for detecting polyPaccumulating bacteria in activated...

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…PolyP is composed of linear phosphate polymers, which can represent .30 mM equivalent Pi while free cytosolic orthophosphate concentrations are 20 mM (Pinson et al 2004). Most polyP is stored in the vacuole, although some is found in other cellular compartments (e.g., nucleus and mitochondria) (Urech et al 1978;Saito et al 2005).…”

Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription

“…Additional investigations of polyP localization on U266 MC were performed using the recombinant polyP-binding domain (PPBD) of E. coli PPX linked to an Xpress epitope tag. 18 Explanations of the method used for PPBD labeling are given in the legend to Online Supplementary Figure S2B. The localization of other proteins was determined by immunostaining, as also described in the legend to Online Supplementary Figure S2B, but using 1/50 dilutions of primaryspecific antibodies and 1/100 dilutions of labeled secondary antibodies (Alexa647-labeled anti-rabbit or Alexa647-labeled antimouse IgG).…”

“…In general, each cell had one to three of these polyP-rich structures with an average diameter of 1.4 μm, and all were located inside the nucleus ( Figure 1A). U266 MC were also stained using a protein that contains a PPBD 18 (Online Supplementary Figure S2B), or with toluidine blue, a polyP-detecting dye 26 (Online Supplementary Figure S2C). In all cases, U266 MC showed staining in intranuclear structures and also in the cytoplasm.…”

Organelle size control – increasing vacuole content activates SNAREs to augment organelle volume through homotypic fusion

“…Their size and copy number are influenced by an equilibrium of membrane fusion and fission (Baars et al, 2007;Efe et al, 2007;Zieger and Mayer, 2012) and by environmental changes (Peters et al, 2004;Weisman, 2003). A crucial advantage is that vacuole content is dominated by only few compounds, polyphosphate ( polyP) and the basic amino acids that associate with it (Saito et al, 2005). PolyP is a polymer of up to hundreds of inorganic phosphate units linked by phosphoanhydride bonds.…”