Distribution of Plasma-membrane Fragments during Zonal Centrifugations of Homogenates from Aerobic Saccharomyces cerevisiae

Abstract

I N T R O D U C T I O NDuring zonal centrifugations of homogenates from glucose-repressed Saccharomyces cerevisiae, the buoyant densities of various plasma-membrane fragments greatly depend on their content of heavy glycoprotein particles (Nurminen, Taskinen & Suomalainen, I 976). Experiments with homogenates from aerobic S. cerevisiae are described here. A complicating factor in the isolation of the yeast plasma membrane is that the only marker enzyme known for it, Mg2+-dependent adenosine triphosphatase (Mg-ATPase), is also present in mitochondria. The mitochondrial Mg-ATPase, however, differs from the Mg-ATPase of the plasma membrane in being oligomycin-sensitive. In aerobic yeast, fully developed mitochondria are readily detectable in electron micrographs and the activity of the typical mitochondrial enzymes is high. Therefore the possible presence of mitochondrial membranes in the preparations of plasma membranes is easier to detect than in the glucose-repressed yeast. M E T H O D SOrganism. This was commercial baker's yeast (Saccharomyces cerevisiae), from the Rajamaki Factories of the Finnish State Alcohol Monopoly (Alko).Preparation qf subcellular fractions. Fresh cells were disrupted in a Mini-mill disintegrator, high-speed zonal centrifugations were performed in a B XV zonal rotor, and several of the membrane fractions obtained by zonal centrifugation on buffered sucrose were further fractionated on gradients of iso-osmotic Urografin as described previously (Nurminen et al., 1976).Biochemical determinations. Enzyme assay procedures and analytical methods were as described by Nurminen et al. (1976).Electron microscopy. Electron micrographs were obtained as described by Nurminen et al.

Two distinct subfractions in isolated Saccharomyces cerevisiae plasma membranes

“…Cells were grown in minimal medium with 2% glucose. species (density, 1.15 and 1.17 g/cm3) are so similar in density that they could not be resolved with the steeper gradients that are described in other reports (24,30,31,38,45). Densities of plasma membranes from previous reports show variable values which aggravate comparisons of different preparations.…”

Association of Divalent Ions With Proteins of the Yeast Plasma Membrane

“…These enzymes could be measured in the protoplast lysate or in bands of the sucrose gradient other than the plasma membrane band. Oligomycin, which inhibits mitochondria1 ATPase of yeast (Nurminen et al 1977), did not affect the ATPase of the preparation and ouabain was not inhibitory. The specific activity of Mg2' -dependent ATPase was 12.7 / .…”

Lipid-mediated glycosylation of endogenous proteins in isolated plasma membrane of Saccharomyces cerevisiae

“…During isolation and purification of the fraction containing plasma membranes from a yeast homogenate by density gradient centrifugation, we have identified the plasma membranes by their specific marker enzyme, the oligomycin-insensitive, Mg2e-dependent ATPase with a pH optimum of 6.5. A similar enzyme is also present in mitochondria (pH optimum, 8 to 9), but it differs from the plasma membrane ATPase in its sensitivity to inhibition by oligomycin (3,16,17). Because data concerning the distribution of membranes during centrifugation on sucrose and Urografim gradients indicate that the buoyant density of mitochondrial membranes is close to that of plasma membranes (16,17), the most important contaminant to be reckoned with would seem to be mitochondrial membrane fragments.…”

“…A similar enzyme is also present in mitochondria (pH optimum, 8 to 9), but it differs from the plasma membrane ATPase in its sensitivity to inhibition by oligomycin (3,16,17). Because data concerning the distribution of membranes during centrifugation on sucrose and Urografim gradients indicate that the buoyant density of mitochondrial membranes is close to that of plasma membranes (16,17), the most important contaminant to be reckoned with would seem to be mitochondrial membrane fragments. We have therefore determined the activity of the Mg2+-ATPase at pH 6.5 in fractions from density gradients in the presence and absence of oligomycin.…”