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Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Growth of Saccharomyces cerevisiae on Ethanol Induces the Synthesis of PolyPV Fraction

The content of five fractions of inorganic polyphosphates (PolyP) in the yeast Saccharomyces cerevisiae grown on glucose or ethanol has been studied. During growth on glucose, when the main pathway of energy metabolism was glycolysis, cells accumulated by 25% more PolyP than at growth on ethanol. However, the content of PolyP V fraction, which is usually present in a negligible amount, substantially increased at high aeration. Under active synthesis of PolyP in ?phosphate overplus? conditions ethanol-grown yeasts synthesized a large amount of PolyP V and its level quantified as 30% of the total PolyP pool. The polyphosphate nature of PolyP V fraction has been shown for the first time by means of 31P-NMR assay. In contrast to other fraction, most of the energy needed for PolyP V synthesis is, apparently, derived from actively functioning mitochondria.

PolyP, a linear polymer of orthophosphate (Pi), plays a significant role in regulation of many biochemical processes (1-3). PolyP can be isolated from cells by different methods (3), but the most complete extraction of these compounds from S. cerevisiae cells occurred during successive treatment of biomass in the cold with 0.5 N HClO4 (acid-soluble fraction, PolyP I), NaClO4 solution (salt-soluble fraction, PolyP II), NaOH, pH 9-10 (alkali-soluble fraction, PolyP III), NaOH, pH 12 (alkali-soluble fraction, PolyP IV), and 0.5 N HClO4, 90° (fraction PolyP V) (4,5).

The change of the content and chain length of PolyP during first four fractions extracted from S. cerevisiae grown on glucose has been well studied. Individual fractions appeared to be different in the rates of both synthesis and utilization. At the same time, the PolyP V fraction was present in a very insignificant quantity of the total PolyP pool, which prevented its strict reliable identification (4). Acid-insoluble PolyP isolated according to Clark et al. (6) were accumulated to a lesser extent at a growth on ethanol than at a growth on glucose (7).

Since different PolyP fractions are metabolically compartmented (1, 3), we studied the content of separate PolyP fractions in the yeast growing on different carbon sources under various aeration. The S. cerevisiae strain VKM Y-1173 was grown on the Reader medium (8) containing 10 mM phosphate, 2% glucose, or 1.5 % ethanol (v/v) to the mid-log growth phase under low and high aeration conditions. PolyP fractions were isolated from cells as described (4, 5).

Total PolyP level at growth on glucose under low aeration was 16.7 mg P/g dry weight. The growth on glucose at higher aeration resulted in a minor decrease of their level (13.3 mg P/g dry weight). The total PolyP during growth on ethanol decreased to 10 mg P/g dry weight, which corresponded to the data by Schuddemat et al. (7). It is worth mentioning that the content of PolyP fractions depended on aeration and carbon source. In all three variants of growth, the PolyP II fraction was almost unchanged, while the level of PolyP V fraction significantly increased in the cells grown either on ethanol or glucose under intensive aeration comparing to the cells grown under weak aeration (Fig. 1). High level of the fraction V at the most intensive cell growth has been observed for the first time.


Figure 1: PolyP fractions in yeast cells in the logarithmic phase of growth: 1, glucose under low aeration. Under high aeration: 2, glucose; 3, ethanol.

In many organisms, including yeasts, the addition of Pi to a culture previously deprived of phosphorus resulted in a rapid accumulation of PolyP to a level many times exceeded that under growth on a complete medium. This phenomenon is called ?hypercompensation? or ?phosphate overplus? (3).

The accumulation of PolyP under hypercompensation in the cells grown at high aeration on ethanol was lower than that on glucose (Fig. 2). However, the level of PolyP V was higher than that at grown on glucose accounting to 30% of the total PolyP pool. By means of 31P-NMR (Fig. 3), for the first time we showed the polyphosphate nature of this fraction.


Figure 2: The content of PolyP fractions in yeast cells in 60 min of phosphate overplus at a growth under intensive aeration on Rider medium: 1, with glucose; 2 , with ethanol. The yeast was grown on a complete medium with phosphate to the beginning of logarithmic growth phase, then transferred to a medium without phosphate, and in 7 h (the PolyP level decreased ca. 7-fold) transferred again to a medium with phosphate.



Figure 3: 31P-NMR spectrum of the PolyP V fraction. 1, core phosphate groups. Residual biomass after successive removal of fractions PolyP I, PolyP II, PolyP III, and PolyP IV was suspended in distilled water, neutralized with 0,1 HCl, and EDTA solution was added to a final concentration of 30 mM. 31P-NMR spectra were recorded on a spectrometer with superconducting magnet operated at 242.9 MHz, 45° pulse, and 1 sec delay. Scan number was 500. The solution of disodium salt of ethylenediaminephosphonic acid with a chemical shift of 12.8 ppm with respect to 85% H3PO4 was used as the standard.

Our data pointed to a suggestion that different PolyP fractions are metabolically associated with different biochemical processes in the cell. Little is still known on mechanism of PolyP synthesis in the yeast cell. Only one enzyme of PolyP biosynthesis, dolichyl-diphosphate: polyphosphate phosphotransferase (EC 2.7.4.20), has been found in S. cerevisiae (3). However, this pathway of PolyP biosynthesis, associated with the synthesis of yeast cells wall, provides accumulation of about 20-30% of total PolyP. Other known synthetic enzymes, polyphosphate kinase and 3-phospho-d-glyceroyl-phosphate:polyphosphate phosphotransferase, have not been revealed in this yeast. Thus, the question of PolyP biosynthesis due to ATP terminal phosphate in S. cerevisiae is still open (3).

It is known that in S. cerevisiae, even under aeration, glucose repressed the synthesis of functional components of several pathways that are not required for glycolytic metabolism of hexoses (e.g., Krebs cycle, respiratory chain and oxidative phosphorylation, glyoxylate shunt and gluconeogenesis) (9).

Apparently, most of the energy needed for polyphosphate synthesis is derived from the glycolytic pathway (1, 3, 7). The synthesis of PolyP V seems to be connected directly to the energy produced by actively functioning mitochondria in contrast to other polyphosphate fractions.

Acknowledgments

This research was supported by the Support Foundation of Leading Scientific Schools of Russia NS-4318.2006.4 and Russian Basic Research Foundation 05-04-48175. The authors are grateful to Professor V. P. Kutyshenko (Institute of Cell Biophysics, RAS) for 31P-NMR-spectroscopy.

References and Footnotes
  1. Kulaev, I. S., Vagabov, V. M. Adv Microbiol Physiol 24, 83-171 (1983).
  2. Kornberg, A., Rao, N. N., Ault-Riche, D. Ann Rev Biochem 68, 89-125 (1999).
  3. Kulaev I., Vagabov, V., Kulakovskaya, T. The Biochemistry of Inorganic Polyphosphates. John Wiley & Sons, Ltd., Chichester, 271 (2004).
  4. Vagabov V. M., L. V. Trilisenko, I. N. Shchipanova, L. A. Sibeldina, I. S. Kulaev. Microbiology (Moscow) 67, 193-198 (1998).
  5. Kulaev I., Vagabov, V., Kulakovskaya, T. J Biosci Bioeng 88, 111-129 (1999).
  6. Clark, J. E., Beegen, H., Wood, H. G. J Bacteriol 168, 1212-1219 (1986).
  7. Schuddemat, J., de Boo, R., Van Leeuwen, C. C. M., Van den Broek, P. J. A., Van Steveninck, J. Biochim Biophys Acta 100, 191-198 (1989).
  8. Reader, V. Biochem J 21, 901-907 (1927).
  9. Gelede R, Van de Velde, S., Van Dijck, P.,Trevelein, J. M. Gen Biol 4, 233.1-233.5 (2003).

Vladimir M. Vagabov
Ludmila V. Trilisenko*
Igor S. Kulaev

Institute of Biochemistry and Physiology of Microorganisms
Russian Academy of Sciences
142290 Pushchino, Russia

*Phone: +7 496 773 2668
Fax: +7 495 956 3370
Email: luvat@rambler.ru