Virtually all of the polyphosphate (PP) present in yeast protoplasts can be recovered in a crude particulate fraction if polybase-induced lysis is used for disrupting the protoplasts. This fraction contains most of the vacuoles, mitochondria and nuclei. Upon the purification of vacuoles the PP is enriched to the same extent as are the vacuolar markers. The amount of PP per vacuole is comparable to the amount of PP per protoplast. The possibility that PP is located in the cell wall is also considered. In the course of the incubation necessary for preparing protoplasts, 20% of the cellular PP is broken down. As this loss of PP occurs to the same extent in the absence of cell wall degrading enzymes, it is inferred that internal PP is metabolically degraded, no PP being located in the cell walls. It is concluded that in Saccharomyces cerevisiae most if not all of the PP is located in the vacuoles, at least under the growth conditions used.
The transport of L-arginine was studied in isolated vacuoles of Sacclzaromycrs cerevisiae. A centrifugation method allowed rapid separation of the fragile vacuoles from the incubation media so that initial uptake rates of [14C]arginine could be measured.Labelled arginine added to the medium was accumulated in the isolated vacuoles; it was found to exchange specifically with the arginine already present in the vacuoles. Such an exchange did not take place in intact spheroplasts.The pH dependence of the arginine transport in the vacuoles was tested. As the vacuoles are unstable in the pH range of optimal transport activity (pH above 7.0), the pH optimum of the transport reaction could not be determined.From the temperature dependence, the apparent energy of activation was calculated to be 9800 call mol.Arginine transport shows saturation kinetics with an apparent K,,, of 30 pM in the isolated vacuoles, and of 1.5 pM in the spheroplasts.Competition experiments with amino acids and arginine analogues demonstrated that the arginine transport in both vacuoles and spheroplasts, is highly specific. The two systems, however, were shown to have distinct specificities.The inhibition of vacuolar L-arginine transport by D-arginine, L-histidine, and L-canavanine was competitive with apparent K , values of 60 pM, 400 pM and 600 pM respectively. In all of these studies, it is taken for granted that the uptake systems investigated are located in the plasmalemma where they mediate the entry of solutes into the cytoplasm, and the possible transport from the cytoplasm into the various membrane-bound compartments within the cell is not considered. However, the structure of eukaryotic cells suggests that transport phenomena associated with the plasmalemma and with intracytoplasmic membrane systems occur sequenAbbreviarion. Mes, 2(N-Morpholino)ethane sulfonic acid. tially, and that the uptake characteristics of intact cells may result from the interdependent action of consecutive transport systems.In the case of amino acids, the question of intracellular transport arose from previous work on the yeast vacuole. The bulk of the soluble amino acids has been found to be located and stored within this cell organelle, separated from the small metabolic pool of amino acids in the cytoplasm [lo, 111. Thus, the uptake of amino acids into yeast cells apparently involves transport both from the medium into the cytoplasm, and from the cytoplasm into the vacuole.Hitherto, attempts to analyse these systems separately by isolating either cell membrane vesicles [I21 or intact vacuoles [13] have failed. The present work gives a first account on a specific amino-acid transport system in isolated vacuoles. A system for arginine transport is characterized and some features are compared with those of the arginine uptake by the spheroplasts from which the vacuoles were isolated.Eur. J. Biochem. 54 (1975)
Membrane preparations from growing regions of pea stems and actively-dividing mouse L-cells form lipid-linked saccharides from GDP-mannose and UDP-N-acetylglucosamine. These lipids have properties which are consistent with those of mono- and di-phosphoryl polyisoprenyl derivatives. In experiments using plant membranes, the monophosphoryl derivative labeled with GDP-(14C) mannose contains mannose only, while the diphosphoryl derivative labeled with the same nucleotide sugar is heterogeneous, containing oligosaccharides corresponding to mannosaccharides of 5, 7, and 9--12 residues. Only the diphosphoryl polyisoprenyl derivatives are labeled with UDP-(14C)glucosamine and these contain predominantly chitobiose and N-acetylglucosamine itself. Unlabeled GDP-mannose added after UDP-N-acetyl-(14C)glucosamine results in the formation of higher lipid-linked oligosaccharides which are apparently the same as those which are labeled with GDP-(14C)mannose alone. Incubation of the membranes with GDP-(14C)mannose in the presence of Mn2+, unlabeled UDP-glucose or unlabeled UDP-N-acetylglucosamine results in marked changes in the accumulation of both the polyisoprenyl monophosphoryl mannose and polyisoprenyl diphosphoryl oligosaccharides. Animal cell membranes synthesise lipid-linked oligosaccharides when incubated with UDP-N-acetylglucosamine and GDP-mannose. These oligosaccharides are similar in size to those synthesised by the plant membranes but their formation is more efficient. The potential roles of these compounds in glycoprotein biosynthesis in both plant and animal tissues is discussed.
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