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Applied and Environmental Microbiology, October 2005, p. 5692-5701, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5692-5701.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Direct Labeling of Polyphosphate at the Ultrastructural Level in Saccharomyces cerevisiae by Using the Affinity of the Polyphosphate Binding Domain of Escherichia coli Exopolyphosphatase

Katsuharu Saito,1,{dagger} Ryo Ohtomo,1 Yukari Kuga-Uetake,2 Toshihiro Aono,3 and Masanori Saito4*

Department of Grassland Ecology, National Institute of Livestock and Grassland Science, 768 Senbonmatsu, Nishinasuno, Tochigi 329-2793, Japan,1 Department of Food Production Science, Faculty of Agriculture, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan,2 Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan,3 Department of Environmental Chemistry, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki 305-8604, Japan4

Received 13 December 2004/ Accepted 29 April 2005

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.

* Corresponding author. Mailing address: Department of Environmental Chemistry, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki 305-8604, Japan. Phone and fax: 81 298 38 8300. E-mail: msaito{at}affrc.go.jp.

{dagger} Present address: CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan, and Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Applied and Environmental Microbiology, October 2005, p. 5692-5701, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5692-5701.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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