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

Cell refractive index has been used to monitor peroxisome behaviour in the yeast Hansenula polymorpha 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 whosesubunits are arranged to form a regular crystalloid. Peroxisomes undergo a degradation process mediated by vacuoles whenever they and theirenzymes become metabolically redundant (e.g. during growth on glucose). Flow cytometric analyses of side scattered light (depending on cellvolume, morphology and structure) and fluorescein isothiocyanate retention (due to the vacuole) were made on two wild-type strains of H. polymorpha during exponential growth in glucose and methanol media and during nutritional shifts from one carbon source to the other. The sameparameters 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.