Detection of a yeast polyphosphate fraction localized outside the plasma membrane by the method of phosphorus-31 nuclear magnetic resonance

Tijssen, J.P.F.; Steveninck, J. Van

doi:10.1016/s0006-291x(84)80269-7

Cited by 25 publications

(9 citation statements)

References 16 publications

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“…Subsequent addition of EDTA to the suspension restored the original spectrum, confirming the localization of a poly-Pi fraction outside the plasma membrane of S. fragilis (31). Membrane-impermeable chelators, such as EDTA, have been used to differentiate between the periplasmic and cytoplasmic pools of poly-Pi in Mycobacterium smegmatis (21).…”

Section: Discussionmentioning

confidence: 77%

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Utilization by Escherichia coli of a high-molecular-weight, linear polyphosphate: roles of phosphatases and pore proteins

Rao

Torriani

1988

J Bacteriol

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We observed that wild-type Escherichia coli utilized a linear polyphosphate with a chain length of 100 phosphate residues (poly-P100) as the sole source of phosphate in growth medium. A mutation in the gene phoA of alkaline phosphatase or phoB, the positive regulatory gene, prevented growth in this medium. Since no alkaline phosphatase activity was detected outside the wild-type cells, the periplasmic presence of the enzyme was necessary for the degradation of polyphosphate. A 90% reduction in the activity of periplasmic acid phosphatase with a pH optimum of 2.5 (AappA mutants) did not affect polyphosphate utilization. Of the porins analyzed (OmpC, OmpF, and PhoE), the phoB-inducible porin PhoE was not essential since its absence did not prevent growth. To study how poly-P100 diffused into the cells, we used high-resolution 31P nuclear magnetic resonance (31P NMR) spectroscopy. The results suggest that poly-PlOO entered the periplasm and remained in equilibrium between the periplasm and the medium. When present individually, porins PhoE and OmpF facilitated a higher permeability for poly-P100 than porin OmpC did. The degradation of polyphosphate by intact cells of E. coli observed by 31P NMR showed a time-dependent increase in cellular phosphate and a decrease in polyphosphate concentration.

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Section: Discussionmentioning

confidence: 77%

“…1C). In vivo high-resolution 31P NMR spectroscopy selectively monitors soluble pools of poly-Pi (5,31). Cytoplasmic or cell surface-associated polyPi complexes are not detected under normal spectral conditions.…”

Section: Discussionmentioning

confidence: 99%

Utilization by Escherichia coli of a high-molecular-weight, linear polyphosphate: roles of phosphatases and pore proteins

Rao

Torriani

1988

J Bacteriol

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“…Because of the small amount of polyP present or its short chain length, our procedure using PPBD might not have detected polyP signals in the mitochondria. Many researchers have described polyP in the cell periphery, the cell envelope, or the cell wall in yeasts (48)(49)(50)54) and E. magnusii (26). However, we could not detect polyP signals in such structures in S. cerevisiae.…”

Section: Discussionmentioning

confidence: 99%

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

Saito

Ohtomo

Kuga-Uetake

et al. 2005

Appl Environ Microbiol

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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...

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“…Histochemical staining methods are not generally able to detect such small amounts of aggregated phosphates. Lead staining results in better visualization of polyphosphates (Tijssen & van Steveninck, 1984), but its use would have complicated the interpretation of the X-ray spectra by generating a peak close to that of P, possibly masking it. During preparation for conventional electron microscopy, phosphates may also be solubilized and washed out (Tijssen, van Steveninck & De Bruijn, 1985).…”

Section: Phosphorylated Fractionsmentioning

confidence: 99%

Polyphosphates in the red macroalga Chondrus crispus (Rhodophyceae)

1997

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SUMMARYPlants of Chondrus crispus Stackhouse, collected from mid-littoral tidepools, were treated as follows. Some plants were kept for a few weeks under controlled starvation conditions in order to decrease their initial content of total tissue phosphorus, then incubated for up to 48 h in phosphorus (15 /^M) and nitrogen (25 /iM)enriched sea water. Other plants were directly incubated in enriched sea water. Chemical analyses showed that the total phosphorus content of fresh and starved plants remained stable, reflecting the nutritional status of the plants. The predominant acid-soluble phosphate fraction was larger in fresh than in starved plants. The content of acid-soluble polyphosphates, similar in both types of plants at the beginning of the experiment, doubled in starved plants, and increased by a factor of 2-7 in fresh plants, over 48 h. The content of acid-insoluble polyphosphates was lower than that of acid-soluble polyphosphates.Transmission electron microscopy and energy dispersive X-ray microanalysis confirmed the presence, mostly in medullary cells, of acid-insoluble polyphosphates in the form of cytoplasmic granules and precipitates along the plasmalemma, particularly near pit plugs. This is the first report of such phosphorus storage structures in a red macroalgal species.

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Detection of a yeast polyphosphate fraction localized outside the plasma membrane by the method of phosphorus-31 nuclear magnetic resonance

Cited by 25 publications

References 16 publications

Utilization by Escherichia coli of a high-molecular-weight, linear polyphosphate: roles of phosphatases and pore proteins

Utilization by Escherichia coli of a high-molecular-weight, linear polyphosphate: roles of phosphatases and pore proteins

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

Polyphosphates in the red macroalga Chondrus crispus (Rhodophyceae)

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