Monitoring of peroxisome induction and degradation by flow cytometric analysis of cells grown in methanol and glucose media: cell volume, refractive index and FITC retention Free

Abstract

Cell refractive index has been used to monitor peroxisome behaviour in the yeast by means of flow cytometry. Peroxisomes are inducible organelles which may occupy a large fraction of the cell volume when yeast cells are growing in methanol media. These organelles harbour a catalase that decomposes the hydrogen peroxide produced in methanol oxidation by alcohol oxidase, a peroxisomal enzyme whose subunits are arranged to form a regular crystalloid. Peroxisomes undergo a degradation process mediated by vacuoles whenever they and their enzymes become metabolically redundant (e.g. during growth on glucose). Flow cytometric analyses of side scattered light (depending on cell volume, morphology and structure) and fluorescein isothiocyanate retention (due to the vacuole) were made on two wild-type strains of during exponential growth in glucose and methanol media and during nutritional shifts from one carbon source to the other. The same parameters were also analysed for a mutant strain only partially repressed by glucose. We show that both the parameters are substrate-dependent and appear to reflect peroxisome development in the cells. The data reported correlate well with the known cytological and biochemical data, showing the possibility of using flow cytometry, a fast and sensitive technique, to analyse the dynamics of peroxisome proliferation and degradation in response to environmental as well as genetic factors.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-140-11-3161
1994-11-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/140/11/mic-140-11-3161.html?itemId=/content/journal/micro/10.1099/13500872-140-11-3161&mimeType=html&fmt=ahah

References

  1. Alberghina L., Porro D. 1993; Quantitative flow cytometry: analysis of protein distributions in budding yeast.. A mini review. Yeast 9:815–823
    [Google Scholar]
  2. Bohmer R.M., King C. N. J. 1984; Flow cytometric analysis of immunogold cell surface label. Cytometry 5:543–549
    [Google Scholar]
  3. Borst P. 1989; Peroxisome biogenesis revisited.. Biochim Biophys Acta 1008:1–13
    [Google Scholar]
  4. De Mesquita G. D. S., Hoopen R. T., Woldringh C. L. 1991; Vacuolar segregation to the bud of Saccharomyces cerevisiae: an analysis of morphology and timing in the cell cycle.. J Gen Microbiol 137:2447–2454
    [Google Scholar]
  5. Douma A. C., Veenhuis M., Suiter G. J., Waterham H. R., Verheyden K., Mannaerts G. P., Harder W. 1989; Permeability properties of peroxisomal membranes from yeast.. Arch Microbiol 153:490–495
    [Google Scholar]
  6. Dubelaar G.B.J., Visser M.J. W., Donze M. 1987; Anomalous behaviour of forward and perpendicular light scattering of a cyanobacterium owing to gas vacuoles.. Cytometry 8:405–411
    [Google Scholar]
  7. Giuseppin M.L.F., Van Eijk, B C Bes H.M. 1988; Molecular regulation of methanol oxidase activity in continuous cultures of Hansenula polymorpha. . Biotechno I Bioeng 32:577–583
    [Google Scholar]
  8. Gleeson M. A., Ortori G. S., Sudbery P. E. 1986; Transformation of the methylotrophic yeast Hansenula polymorpha . J Gen Microbiol 132:3459–3465
    [Google Scholar]
  9. van der Klei I. J., Bystrykh L. V., Harder W. 1990; Alcohol oxidase from Hansenula polymorphaCBS4732.. Methods Enyymol 188:420–427
    [Google Scholar]
  10. van der Klei I. J., Harder W., Veenhuis M. 1991; Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeasts: a review. Yeast 7:195–209
    [Google Scholar]
  11. Lazarow P.B., Moser H. W. 1989; Disorders of peroxisome biogenesis.. In The Metabolic Basis of Inherited Diseases , 6th edn. pp. 1479–1509 Edited by Scriver C. R., Beaudet A. L. W., Sly S., Valle D. New York: McGraw-Hill Publications;
    [Google Scholar]
  12. Martegani E., Porro D., Ranzi B. M., Alberghina L. 1990; Involvement of a cell size control mechanism in the induction and maintenance of oscillations in continuous cultures of budding yeast.. Biotechnol Bioeng 36:453–459
    [Google Scholar]
  13. Moser H. W., Bergin A., Cornblath D. 1991; Peroxisomal disorders.. Can J Biochem Cell Biol 69:463–474
    [Google Scholar]
  14. Salzman G. C., Singham S. B., Johnston R. G., Bohren C. F. 1990; Flow Cytometry and Sorting. , 2nd edn. pp. 81–107 Edited by Melamed M. R., Lindmo T., Mendelsohn M. L. . New York: Wiley-Liss;
    [Google Scholar]
  15. Shapiro H.M. 1988; Practical Flow Cytometry. , 2 nd edn. New York: Alan R. Liss;
    [Google Scholar]
  16. Srienc F., Arnold B., Bailey J. E. 1984; Characterization of intracellular accumulation of poly-yS-hydroxybutyrate (PHB) in individual cells of Alcaligenes eutrophusH16 by flow cytometry.. Biotechnol Bioeng 26:982–987
    [Google Scholar]
  17. Veenhuis M., Harder W. 1991; Microbodies. In The Yeasts , 2nd edn. 4: pp. 601–653 London: Academic Press;
    [Google Scholar]
  18. Veenhuis M., Zwart K. B., Harder W. 1978; Degradation of peroxisomes after transfer of methanol-grown Hansenula polymorpha into glucose-containing media. FEBS Microbiol Lett 4:283–286
    [Google Scholar]
  19. Veenhuis M., Douma A., Harder W., Osumi M. 1983a; Degradation and turnover of peroxisomes in the yeast Hansenula polymorphainduced by selective inactivation of peroxisomal enzymes. Arch Microbiol 134:193–201
    [Google Scholar]
  20. Veenhuis M., van Dijken J.P., Harder W. 1983b; The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts.. In Advances in Microbial Physiology pp. 1–82 Edited by Rose H. J., Gareth Morris A., Tempest D. W. London : Academic Press;
    [Google Scholar]
  21. Waterham H. R., Titorenko V. I., Swaving G. J., Harder W., Veenhuis M. 1993; Peroxisomes in the methylotrophic yeast Hansenula polymorphado not necessarily derive from pre-existing organelles.. EMBO J 12:4785–4794
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-140-11-3161
Loading
/content/journal/micro/10.1099/13500872-140-11-3161
Loading

Data & Media loading...

Most cited Most Cited RSS feed