Graduate Thesis Or Dissertation
 

The distribution and influence of sterols in yeast membranes

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  • Plasma membranes from Saccharomyces cerevisiae were prepared by a new procedure involving lyticase treatment of the yeast cells. The plasma membranes were right-side out, closed vesicles of uniform appearance with a sterol to phospholipid molar ratio of 0.365. The thermotropic behavior of these plasma membranes from wild-type yeast and from its sterol mutants was examined by differential scanning calorimetry, fluorescence polarization, nuclear magnetic resonance, and Arrhenius kinetics of plasma membrane enzymes. While DSC failed to demonstrate any lipid transition, fluorescence anisotropy data indicated that lipid transitions were occurring in the plasma membranes of the sterol mutants but not the sterol wild-type. Physical studies of mitochondria of the yeast wild-type and its sterol mutants gave similar results. Parallel experiments with model membrane liposomes verified that the phase transition observed by fluorescence polarization is dependent on the sterol present. NMR data suggested that the plasma membranes from both types of yeast were undergoing a phase transition, but the nature of this transition was undefined. The apparent discrepancies in these results may be ascribed to lateral phase separations of the phospholipid and sterol present in the membranes. The temperature dependence of the plasma membrane enzymes, chitin synthetase and Mg⁺⁺-ATPase, was also investigated. The Arrhenius kinetics of chitin synthetase did not reveal any transitions in either the sterol mutant or wild-type plasma membranes, yet the Arrhenius kinetics of the Mg⁺⁺-ATPase suggested that lipid transitions were occurring in both cases. To explain this phenomenon, a model is proposed involving the existence of at least two different domains in the yeast plasma membrane, one domain being sterol-rich and the other domain being sterol-poor.
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