Polybase induced lysis of yeast spheroplasts

Dürr, Mathias; Böller, Thomas; Wiemken, Andres

doi:10.1007/bf00447152

Cited by 94 publications

(11 citation statements)

References 29 publications

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“…In this respect, the grape skin tissue closely resembles other, high sugar-containing, tissues such as beet roots, which also require high osmolarity medium for the production of stable protoplasts and vacuoles (14). The protoplasts produced from the grape skin did not release intact vacuoles upon treatment with 0.2 M Na4P207-HCl (pH 8.0) (26), upon dilution of the osmoticum (20), by polybasic DEAE-dextran treatment (10), or by shearing forces created upon uptake and expulsion in a syringe (18).…”

Section: Resultsmentioning

confidence: 97%

Vacuolar Contents of Fruit Subepidermal Cells from Vitis Species

Moskowitz¹,

Hrazdina²

1981

Plant Physiol.

118

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termination of phenolic compounds, sugars, acids, and cations, the skins were removed manually from the grapes, rinsed briefly with distilled H20, frozen in N2, and freeze-dried. To prevent oxidation of phenolic compounds, vacuum was released with N2, and samples were stored in N2 atmosphere., Preparation of Vacuoles. Grape berries (1 kg) were rinsed briefly in 70%o aqueous ethanol and dried on paper towels, and the skins were manually removed by gentle squeezing and cut in half. Batches of about 30 g of skins were rinsed with 100 ml buffer (17 mm citric acid, 70 mim Na2HPO4, and 1.2 M sorbitol at pH 6.0) for 30 min in 1,000-ml beakers on a reciprocal shaker (100 rpm). The buffer was replaced with a Cellulysin (1.5%; Calbiochem) and Macerase (1%; Calbiochem) solution in the same volume as the above buffer. The grape skins were digested for 7 h at room temperature, the incubation medium decanted, and the protoplasts sedimented at 100g in a centrifuge. The sediment was resuspended in the rinsing buffer from above (1,500 ml) and filtered through a glass wool layer in a Buchner funnel under mild suction (aspirator). This treatment produced vacuoles from protoplasts. The vacuoles were sedimented at 100g, resuspended in 225 ml rinsing buffer, and sedimented by gravity twice. The number of vacuoles in a preparation was determined in a well slide using 5 ,Al aliquot and 20 ul. buffer.Disruption of Vacuoles. The vacuole preparations were transferred into 40-ml centrifuge tubes and the vacuoles sedimented at 50g. The supernatant buffer was decanted, the volume made up to 40 ml with distilled H20, and the preparation homogenized by sonication. Microscopic examination of this preparation showed that all vacuoles had lysed. The homogenate was centrifuged at 180,000g for 2 h to remove membranous material. The supernatant (37 ml) was decanted, frozen in N2, and stored at -40 C.Identification of Anthocyanins. The anthocyanins in DeChaunac grapes have been previously identified (28).Determination of Total Anthocyanin Content in Grape Skins.Freeze-dried grape skin powder (1.0 g) was extracted with 0.1% aqueous HCI (100 ml), the absorbance of this solution measured at 520 nm, and the anthocyanin concentration determined using E = 33,000 1-mol-1cm-' (27).Determination of the Total Anthocyanin Content in Vacuole Preparations. One ml of the vacuole lysate was used for the determination, as described above.Quantitative Determination of the Individual Anthocyanins. The individual anthocyanin content of grape skins and vacuole preparations was determined by quantitative adaptation ofa previously described HPLC method (28). Only pigments present in significant amounts were quantitated. For the establishment of anthocyanin standard curves, the following pigment stock solutions were pre-

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Section: Resultsmentioning

confidence: 97%

Vacuolar Contents of Fruit Subepidermal Cells from Vitis Species

Moskowitz¹,

Hrazdina²

1981

Plant Physiol.

118

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“…In some batches (Table S1), TCEP-HCl (Tris(2-carboxyethyl)phosphine, Thermo Scientific, #20491; 5 mM final concentration) was used instead of dithiothreitol (DTT, 10 mM final). Cells were disrupted using 100 µg/ml of diethylaminoethyl dextran (DEAE-Dextran, Sigma-Aldrich) in 15% Ficoll (Fisher Bioreagents) buffer (22). The resulting cell lysate was subjected to density gradient centrifugation (Beckman Coulter Optima L-90K ultracentrifuge, Sw-32 Ti rotor).…”

Section: Methodsmentioning

confidence: 99%

Mössbauer and EPR Study of Iron in Vacuoles from Fermenting Saccharomyces cerevisiae

et al. 2011

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Vacuoles were isolated from fermenting yeast cells grown on minimal medium supplemented with 40 µM 57Fe. Absolute concentrations of Fe, Cu, Zn, Mn, Ca, and P in isolated vacuoles were determined by ICP-MS. Mössbauer spectra of isolated vacuoles were dominated by two spectral features; a mononuclear magnetically isolated high-spin (HS) FeIII species coordinated primarily by hard/ionic (mostly or exclusively oxygen) ligands, and superparamagnetic FeIII oxyhydroxo nanoparticles. EPR spectra of isolated vacuoles exhibited a gave ~ 4.3 signal typical of HS FeIII with E/D ~ 1/3. Chemical reduction of the HS FeIII species was possible, affording a Mössbauer quadrupole doublet with parameters consistent with O/N ligation. Vacuolar spectral features were present in whole fermenting yeast cells; however, quantitative comparisons indicated that Fe leaches out of vacuoles during isolation. The in vivo vacuolar Fe concentration was estimated to be ~1.2 mM while the Fe concentration of isolated vacuoles was ~220 µM. Mössbauer analysis of FeIII polyphosphate exhibited properties similar to those of vacuolar Fe. At the vacuolar pH of 5, FeIII polyphosphate was magnetically isolated, while at pH 7, it formed nanoparticles. This pH-dependent conversion was reversible. FeIII polyphosphate could also be reduced to the FeII state, affording similar Mössbauer parameters to that of reduced vacuolar Fe. These results are insufficient to identify the exact coordination environment of the FeIII species in vacuoles, but they suggest a complex closely related to FeIII polyphosphate. A model for Fe trafficking into/out of yeast vacuoles is proposed.

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“…Lysis of spheroplasts was induced by addition of DEAEdextran to 30 Ag/100 OD6N units of cells (16). Vacuoles were resolved on a discontinuous Ficoll step gradient (4) with the one modification that an extra 4% Ficoll step was added.…”

Section: Methodsmentioning

confidence: 99%

Isolation of yeast mutants defective in protein targeting to the vacuole.

Bankaitis¹,

Johnson²,

Emr³

1986

Proc. Natl. Acad. Sci. U.S.A.

364

309

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We have constructed a PRCI-SUC2 gene fusion that directs the synthesis in Saccharomyces cerevisiae of a hybrid polypeptide consisting of a 433-residue amino-terminal domain derived from the yeast vacuolar protease carboxypeptidase Y (CPY; EC 3.4.16.1) and a 511-residue carboxyl-terminal domain derived from the secreted yeast enzyme invertase (EC 3.2.1.26). Fractionation data indicated that this amount of CPY primary sequence is sufficient to quantitatively divert invertase to the yeast vacuole. The phenotypic consequence of localizing active invertase to the vacuole has enabled us to select for mutants that "mislocalize" the hybrid protein to the cell surface. The corresponding mutations that lead to this effect are all trans-acting and recessive, and they define at least eight complementation groups. These vacuolar protein targeting (vpt) mutants also exhibit hybrid protein independent defects in wild-type CPY delivery to the yeast vacuole. Precursor forms of CPY accumulate in the mutants and are secreted into the yeast periplasm and extracellular medium. The vpt mutants should provide useful information pertaining to the mechanisms by which yeast cells regulate vacuolar protein traffic.

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Polybase induced lysis of yeast spheroplasts

Cited by 94 publications

References 29 publications

Vacuolar Contents of Fruit Subepidermal Cells from Vitis Species

Vacuolar Contents of Fruit Subepidermal Cells from Vitis Species

Mössbauer and EPR Study of Iron in Vacuoles from Fermenting Saccharomyces cerevisiae

Isolation of yeast mutants defective in protein targeting to the vacuole.

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