Skip to main content
Log in

The shock of vacuolar PrA on glycolytic flux, oxidative phosphorylation, and cell morphology by industrial Saccharomyces cerevisiae WZ65

  • Original Paper
  • Published:
European Food Research and Technology Aims and scope Submit manuscript

Abstract

Proteinase A (PrA) is one of the most significant vacuolar proteinase in S. cerevisiae, and it plays an important role in S. cerevisiae physiology and metabolism, especially under unfavorable environment. In this study, the differences in pyruvate kinase (PYK) level under fructose-1,6-diphosphate (FDP) induction and ATP synthesis block among SC1 (the wild-type yeast that was industrial Saccharomyces cerevisiae WZ65), SC2 (PEP4 partial deletion) and SC3 (PEP4 complete deletion) were examined. Results showed that the induction caused by FDP clearly increased PYK expression no matter for which strain, but the increasing effect is more significant for SC2 (P < 0.05). The comparative results of intracellular ATP accumulation showed that the induction by FDP may be affected at the presence of PrA. The block experiment of ATP synthesis showed that PYK activities in PEP4-modified strains are lower than that of the wild type, but the intracellular ATP levels in the wild-type one are generally higher than the PEP4-modified strains after rotenone treatment (P < 0.01). This implies that the effect of PrA deficiency on intracellular ATP accumulation was much more pronounced than the effect of rotenone on oxidative phosphorylation. The cell morphology of three strains was comparatively examined by means of transmission electron microscopy (TEM). The PEP4-modified strains possessed more vacuoles, and cell structure were more integrated than the wild-type strain. Current data preliminarily indicated that the deletion of PEP4 gene in industrial S. cerevisiae WZ65 may not only affected PYK expression but also modulated the oxidative phosphorylation flux.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

SC1:

Industrial Saccharomyces cerevisiae WZ65 (wild-type yeast)

SC2:

Saccharomyces cerevisiae strain with PEP4 partial deletion

SC3:

Saccharomyces cerevisiae strain with PEP4 complete deletion

PrA:

Proteinase A

PrB:

Proteinase B

PEP4 :

A kind of gene that encodes proteinase A

CYP:

Carboxypeptidase

FDP:

Fructose-1,6-diphosphate

HK:

Hexokinase

PFK:

Phosphofructokinase

PYK:

Pyruvate kinase

ATP:

Adenosine triphosphate

ADP:

Adenosine diphosphate

NADH:

Reduced form of nicotinamide-adenine dinucleotide

TEM:

Transmission electron microscopy

GCR%:

Glucose consumption rate, it is the D-value between the original glucose (g/50 mL) in the culturing medium and the residual glucose in the broth (g/50 mL) after certain culturing time divided by the original glucose in the culturing medium (g/50 mL)

References

  1. Parr CL, Keates RA, Bryksa BC, Ogawa M, Yada RY (2007) The structure and function of Saccharomyces cerevisiae proteinase A. Yeast 24:467–480

    Article  CAS  Google Scholar 

  2. Wang ZY, He GQ, Liu ZS, Ruan H, Chen QH, Xiong HP (2005) Purification of yeast proteinase A from fresh beer and its specificity on foam proteins. Int J Food Sci Technol 40(8):835–840

    Article  Google Scholar 

  3. Fegner R, Mahé Y, Pandjaitan R, Kuchler K (1995) Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae. Mol Cell Biol 15(11):5879–5887

    Google Scholar 

  4. Zhang Q, Chen QH, Fu ML, Wang JL, Zhang HB, He GQ (2008) Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination. J Zhejiang Univ Sci B 9(7):527–535

    Article  CAS  Google Scholar 

  5. Zhang HB, Zhang HF, Chen QH, Ruan H, Fu ML, He GQ (2009) Effects of proteinase A on cultivation and viability characteristics of industrial Saccharomyces cerevisiae WZ65. J Zhejiang Univ Sci B 9(7):769–776

    Article  Google Scholar 

  6. Marques M, Mojzita D, Amorim MA, Almeida T, Hohmann S, Moradas-Ferreira P, Costa V (2006) The Pep4p vacuolar proteinase contributes to the turnover of oxidized proteins but PEP4 overexpression is not sufficient to increase chronological lifespan in Saccharomyces cerevisiae. Microbiology 152:3595–3605

    Article  CAS  Google Scholar 

  7. Jurica MS, Mesecar A, Heath PJ, Shi WX, Nowak T, Stoddard BL (1998) The allosteric regulation of pyruvate kinase by fructose-1, 6-bisphosphate. Structure 15(6):195–210

    Article  Google Scholar 

  8. Allert S, Ernes I, Poliszczak A, Opperdoes FR, Michels PA (1991) Molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of Trypanosoma brucei. Eur J Biochem 200:19–27

    Article  CAS  Google Scholar 

  9. Portela P, Howell S, Moreno S, Rossi S (2002) In vivo and in vitro phosphorylation of two isoforms of yeast pyruvate kinase by protein kinase A. J Biol Chem 277:30477–30487

    Article  CAS  Google Scholar 

  10. Clive JB, Brian W, Rodney VB (1971) Regulation of pyruvate pinase by fructose 1, 6-diphosphate in Saccharomyces cerevisiae. Eur J Biochem 18:64–69

    Google Scholar 

  11. Muñoza Ma E, Ponce E (2003) Pyruvate kinase: current status of regulatory and functional properties. Compar Biochem Physiol Part B: Biochem Mol Biol 135(2):197–218

    Article  Google Scholar 

  12. Fell D (1997) Understanding the control of metabolism. Portland Press, London

    Google Scholar 

  13. Schaaff I, Heinisch J, Zimmermann FK (2004) Overproduction of glycolytic enzymes in yeast. Yeast 5(4):285–290

    Article  Google Scholar 

  14. Ruijter GJ, Panneman H, Visser J (1997) Overexpression of phosphofructokinase and pyruvate kinase in citric acid-producing Aspergillus niger. Biochim Biophys Acta 1334:317–326

    CAS  Google Scholar 

  15. Ramos A, Neves AR, Ventura R, Maycock C, López P, Santos H (2004) Effect of pyruvate kinase overproduction on glucose metabolism of Lactococcus lactis. Microbiology 150:1103–1111

    Article  CAS  Google Scholar 

  16. Hearn EM, Patel DR, van den Berg B (2008) Outer-membrane transport of aromatic hydrocarbons as a first step in biodegradation. Proc Natl Acad Sci USA 105:8601–8606

    Article  CAS  Google Scholar 

  17. Larsson C, Nilsson A, Blomberg A, Gustafsson L (1997) Glycolytic flux is conditionally correlated with ATP concentration in Saccharomyces cerevisiae: a chemostat study under carbon- or nitrogen-limiting conditions. J Bacteriol 179:7243–7250

    CAS  Google Scholar 

  18. Liu LM, Li Y, Li HZ, Chen J (2006) Significant increase of glycolytic flux in Torulopsis glabrata by inhibition of oxidative phosphorylation. FEMS Yeast Res 6:1117–1129

    Article  CAS  Google Scholar 

  19. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–427

    Article  CAS  Google Scholar 

  20. Chen QH, Liu XJ, Fu ML, Zhang HB (2010) Effect of PrA encoding gene-PEP4 deletion in industrial S. cerevisiae WZ65 on key enzymes in relation to the glycolytic pathway. Eur Food Res Technol 231(6):943–950

    Article  CAS  Google Scholar 

  21. Davies SEC, Brindle KM (1992) Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae. Biochem 31:4729–4735

    Article  CAS  Google Scholar 

  22. Ronner P, Friel E, Czerniawski K, Fränkle S (1999) Luminometric assay of ATP, phosphocreatine, and creatine for estimation of free ADP and free ATP. Anal Biochem 275(2):208–216

    Article  CAS  Google Scholar 

  23. Yang H, Ren Q, Zhang Z (2006) Chromosome or chromatin condensation leads to meiosis or apoptosis in stationary yeast (Saccharomyces cerevisiae) cells. FEMS Yeast Res 8(6):1254–1263

    Article  Google Scholar 

  24. Adah A, Benghuzzi H, Tucci M, Huang D, Franklin L, Adah F (2006) Metabolic effects of fructose 1,6-bisphosphate in normoxic and hypoxic states of MG63 osteosarcoma cells. Biomed Sci Instrum 42:120–125

    CAS  Google Scholar 

  25. Hunsley JR, Swelter CH (1969) Yeast pyruvate kinase. J Biol Chem 244:4819–4822

    CAS  Google Scholar 

  26. Barwell CJ, Woodward B, Brunt RV (1971) Regulation of pyruvate kinase by fructose 1,6-diphosphate in Saccharomyces cerevisiae. Eur J Biochem 18(1):59–64

    Article  CAS  Google Scholar 

  27. Kazuo S, Yukinar Y, Toshiyuki H, Toshiyuki O (2000) On-line measurement of intracellular ATP of Saccharomyces cerevisiae and pyruvate during sake mashing. J Biosci Bioeng 3(90):294–301

    Google Scholar 

  28. Ziegelhoffer T, Lopez BP, Craig EA (1995) The dissociation of ATP from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydj1p and peptide substrates. J Biol Chem 18(270):10412–10419

    Google Scholar 

  29. Cronwright GR, Rohwer JM, Prior BA (2002) Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae. Appl Environ Microbiol 68(9):4448–4456

    Article  CAS  Google Scholar 

  30. Reibstein D, den Hollander JA, Pilkis SJ, Shulman RG (1986) Studies on the regulation of yeast phosphofructo-1-kinase: its role in aerobic and anaerobic glycolysis. Biochemistry 25:219–227

    Article  CAS  Google Scholar 

  31. Beauvoit B, Rigoulet M, Bunoust O, Raffard G, Canioni P, Guérin B (1993) Interactions between glucose metabolism and oxidative phosphorylations on respiratory-competent Saccharomyces cerevisiae cells. Eur J Biochem 214:163–172

    Article  CAS  Google Scholar 

  32. Senior AE (1988) ATP synthesis by oxidative phosphorylation. Physiol Rev 68:177–231

    CAS  Google Scholar 

  33. Larsson C, Pahlman IL, Gustafsson L (2000) The importance of ATP as a regulator of glycolytic flux in Saccharomyces cerevisiae. Yeast 16:797–809

    Article  CAS  Google Scholar 

  34. Torres NV (1994) Modeling approaches to control of carbohydrate metabolism during citric acid accumulation by Aspergillus niger: II. Sensitivity analysis. Biotechnol Bioeng 44:112–118

    Article  CAS  Google Scholar 

  35. Zhou JW, Liu LM, Shi ZP, Du GC, Chen J (2009) ATP in current biotechnology: regulation, applications and perspectives. Biotechnol Adv 27:94–101

    Google Scholar 

  36. Teichert U, Mechler B, Muller H, Wolf DH (1989) Lysosomal (vacuolar) proteinases of yeast are essential catalysts for protein degradation, differentiation, and cell survival. J Biol Chem 264:16037–16045

    CAS  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the National Hi-Tech Research and Development Program (863) of China (No. 2007AA10Z315).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Qi-He Chen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, XJ., Feng, Y., Fu, ML. et al. The shock of vacuolar PrA on glycolytic flux, oxidative phosphorylation, and cell morphology by industrial Saccharomyces cerevisiae WZ65. Eur Food Res Technol 233, 941–949 (2011). https://doi.org/10.1007/s00217-011-1586-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00217-011-1586-6

Keywords

Navigation