The subcellular location of hexose diphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase in baker's yeast (Saccharomyces cerevisiae) was investigated by density gradient centrifugation of spheroplast lysates obtained by osmotic shock treatment of spheroplasts and centrifugation for 10000 g x min. On the evidence obtained from zonal separations these three enzymes of gluconeogenesis are most probably located in the soluble cytosol.

1. The distributions of several enzymes and other marker components were examined after zonal centrifugations of whole homogenates from glucose-repressed Saccharomyces cerevisiae on sucrose and iso-osmotic Ficoll, and the composition and morphology of the fractions were investigated. 2. After high-speed zonal centrifugation most of the protein, acid and alkaline phosphatases, alkaline pyrophosphatase, adenosine monophosphatase, beta-fructofuranosidase, alpha-mannosidase, NADPH-cytochrome c oxidoreductase and an appreciable amount of phospholipid and sterol were non-sedimentable, i.e. were at densities below 1.09 (g/cm3). Most of the RNA was at p=1.06-1.08 in Ficoll and at p=1.09-1.11 in sucrose. 3. The bulk of the Mg2+-dependent adenosine triphosphatase (Mg-ATPase) was coincident with the main peak of phospholipid and sterol, at median density 1.10, which was also rich in smooth-membrane vesicles. In Ficoll, a minor peak of phospholipid and sterol at p-1.12-1.15 contained a smaller part of the oligomycin-insensitive Mg-ATPase and heavy membrane fragments. In sucrose, several minor peaks of Mg-ATPase were in the mitochondrial density range, and a peak of oligomycin-insensitive Mg-ATPase coincident with a minor peak of phospholipid and sterol at around p-1.25 contained heavy membrane fragments of high carbohydrate content, especially mannose. 4. Further purification of the oligomycin-insensitive Mg-ATPase containing membrane preparations was performed on Urografin gradients. 5. It is argued that the oligomycin-insensitive Mg-ATPase containing membranes are fragments of the plasma membrane, but have different densities because they contain different amounts of glycoprotein particles.

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.

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