Membrane changes in yeast cells caused by sulfhydryl reagents and accompanied by a selective release of sugar

Summary Iodoacetic acid or N-ethylmaleimide included in cell suspensions during measurements of sorbose exit from yeast cells caused sorbose efflux to occur at a uniform rate in contrast to the usual two-phase exit. Cells pretreated with these agents were still capable of sugar uptake, but the entire efflux now occurred at the usual initial rate. Microscopically, the vacuoles of treated cells were observed to be altered or disrupted. Vacuolar effects occurred before methylene blue was able to penetrate the external cell membrane and stain the cells. Vacuoleless cells also allowed a single rate of sorbose efflux. The selective effect upon intracellular membranes is interpreted as a disruption of the boundaries of an internal sugar compartment with the result that sugar exits from the cell at a rate controlled only by the external membrane.     

Stability of the Plasma Membrane in Saccharomyces rouxii and Its Relationship to Glucose Tolerance

The stability of spheroplasts from the osmotrophic yeast Saccharomyces rouxii was studied in buffered solutions of mannitol and glucose. The plasma membranes from cells grown in high glucose concentrations were more stable to osmotic lysis than were membranes from cells grown in lower glucose concentrations. Mannitol was a better osmotic stabilizer than glucose, except when the cells were grown in a high glucose concentration. Spheroplasts from a glucose tolerant-deficient mutant were much less stable than the corresponding spheroplasts from the parent strain, especially when suspended in glucose solutions. These results suggest an involvement of the plasma membrane in the glucose-tolerant mechanism of S. rouxii.     

Increased rates of sugar transport in Saccharomyces cerevisiae a result of sugar metabolism

Preincubation of yeast cells with glucose or other metabolic energy sources increased the rate of sorbose efflux 2- or 3-fold. Stimulated rates persisted for several h, decreasing slowly. They were approximately halved by including K_m concentrations of highly competitive sugars such as deoxyglucose, glucose, fructose and mannose in sorbose efflux suspensions, and were greatly slowed at reduced temperatures. Inhibitors of energy metabolism blocked the rate stimulation, as did cycloheximide; added nitrogen sources increased the rate additionally. The rate of sorbose uptake was also increased, whereas that of dimethylsulfoxide, which enters the cell by simple diffusion, was not changed. Transport of arabinose and fucose also occurred at increased rates. The data indicate a change in the sorbose transport system rather than in membrane permeability. The change, apparently the synthesis of a transport system component, requires metabolic energy and involves protein synthesis.     

Physical Properties of Escherichia coli Spheroplast Membranes

We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained.     

Isolation of Polyribosomes from Yeast During Sporulation and Vegetative Growth

Exponentially growing and sporulating cells of Saccharomyces cerevisiae have been subjected to a variety of conditions which mechanically disrupt the cell in an effort to establish conditions which permit the recovery of intact polyribosomes. Grinding cells for 10 s with glass beads in a Bronwill cell homogenizer was sufficiently gentle to yield a polyribosome content in exponentially growing cells which was similar to values obtained from yeast spheroplasts. Polyribosome patterns in sporulating yeast were similar to those from exponentially growing cells. This technique is fast, reproducible over a wide range of cell concentrations, and eliminates the need to make spheroplasts to recover intact polyribosomes.     

Osmotic Properties of Spheroplasts from Saccharomyces cerevisiae Grown at Different Temperatures

Spheroplasts were prepared from cells of Saccharomyces cerevisiae NCYC 366, grown at 30 or 15 C, by incubating cells with snail-gut juice after pretreatment with 2-mercaptoethanol. Walls of cells grown batchwise or in continuous culture at 15 C were more resistant to digestion with snail juice than walls on cells grown under the same conditions as 30 C. Spheroplasts lysed when suspended in hypotonic solutions of mannitol. The resistance of spheroplasts to osmotic lysis tended to increase when the test temperature was lowered below 30 C. The increased resistance was greater with spheroplasts from cells grown at 15 C. Cations, especially Ca²⁺, protected spheroplasts against osmotic lysis. In general, the protective effects, measured at 30 C, were smaller with spheroplasts from cells grown at 15 C compared with 30 C. Citrate and ethylenediaminetetraacetate (EDTA) decreased the resistance of spheroplasts to osmotic lysis. On the whole, the decrease was greater with spheroplasts from cells grown at 30 C rather than 15 C. In the presence of EDTA, spheroplasts from cells grown at 30 C were less resistant to osmotic lysis at 5 C than at 30 C; when spheroplasts from cells grown at 15 C were similarly examined, they were more resistant to lysis at 5 C than at 30 C. Spheroplast membranes from cells grown at 15 C had slightly but significantly greater contents of Mg²⁺, Ca²⁺, K⁺, and Na⁺ compared with spheroplast membranes from cells grown at 15 C. Mg²⁺ and Ca²⁺ were more easily extracted with EDTA from membranes of 30 C-grown cells than from 15 C-grown cells.     

A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.

We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY). Yeast spheroplasts are lysed by extrusion through polycarbonate filters. After differential centrifugation, a 125,000-g pellet is enriched for radiolabeled proCPY and is used as "donor" membranes. A 15,000-g pellet, harvested from nonradiolabeled cells and enriched for vacuoles, is used as "acceptor" membranes. When these membranes are incubated together with ATP and cytosolic extracts, approximately 50% of the radiolabeled proCPY is processed to mature CPY. Maturation was inhibited by dilution of donor and acceptor membranes during incubation, showed a 15-min lag period, and was temperature sensitive. Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from a PEP4 yeast strain, indicating intercompartmental transfer. Cytosol made from a yeast strain deleted for the VPS33 gene was less efficient at driving transport. Moreover, antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays. This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of a VPS gene product.     

l-Iditol production from l-sorbose by a methanol yeast,Candida boidinii (Kloeckera sp.) No. 2201

Production of l-iditol (iditol) from l-sorbose (sorbose) with d-sorbitol dehydrogenase coupled with NADH regeneration under methanol oxidation was studied with the resting cell system of a methanol yeast, Candida boidinii (Kloeckera sp.) no. 2201.Maximum activities of iditol production and enzymes concerned with the production were found in cells grown on a medium containing methanol and d-xylose (xylose). The highest amount of iditol, 142–148 g/l (94–98% conversion rate), was obtained from 150 g/l of sorbose in the presence of 0.5 M methanol at pH 6.5. Intermittent control of the pH during the cell reaction gave a significantly higher amount of iditol than that obtained without such control. Freeze-thawing cells showed rather higher productivity than resting cells. The product was identified as iditol without contamination of its C-2 epimer, d-sorbitol, by high performance liquid chromatography.     

Correction

We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained.     

Metabolism of [14C]glucose by regenerating spheroplasts of Candida albicans

Spheroplasts of Candida albicans were regenerated in [14C]glucose and buffered magnesium sulphate (0.1 M-Tris/HCl; 0.5 M-MgSO4, pH 7.2) at 35 degrees C. Uptake of glucose by spheroplasts was faster than that by intact yeast cells. After 6 h, 65% of the glucose taken up by the yeast appeared as CO2 and 30% was incorporated into the cellular material. With spheroplasts, 55% of the glucose taken up was expired as CO2, 25% was excreted into the medium as other metabolites and 20% was incorporated into the cells. The regenerating spheroplasts excreted 14C-labelled carbohydrates into the medium which were fractionated on a Sephadex G-15 column. Acid hydrolysis of the low molecular-weight fraction yielded the following sugars: mannose (75.7%), fucose (3.8%), arabinose (3%), galactose (2.1%) and an unidentified monosaccharide (14%). Spheroplasts did not incorporate mannoprotein into the regenerated wall. The wall carbohydrate from regenerated spheroplasts was fractionated on the basis of solubility in sodium hydroxide. The alkali-insoluble fraction was analysed by sequential enzyme hydrolysis; 40% of the incorporated counts were associated with beta (1----3)-linked glucan and 50% with a mixed glucan comprising beta (1----3)- and beta (1----6)-linkages and chitin.     

Kinetic and Morphological Observations on Saccharomyces cerevisiae During Spheroplast Formation

A strain of Saccharomyces cerevisiae which produced elongated cells under our growth conditions was investigated. By digestion of the cell walls with snail enzyme, the cells became spheroplasts after a transient state which we termed "prospheroplast." The prospheroplast could be lysed like the spheroplast, but it retained the shape of the original yeast cell if osmotically protected. Prospheroplasts and spheroplasts were prepared, and thin sections of samples taken throughout the process of wall removal were studied in the electron microscope, at regular intervals up to the time of complete conversion to spheroplasts. In addition, cell wall remnants recovered from spheroplast preparations were shadow cast for electron microscopy. This material revealed structures resembling bud scars with attached membranous matter. The kinetic studies showed that after a certain period of time all cells were transformed into prospheroplasts, whereas spheroplast formation started later, depending on the enzyme concentration. In sections, the prospheroplasts appeared to be formed by detachment of the cell walls. Both the prospheroplasts and the spheroplasts showed asymmetric cytoplasmic membranes in which the outer leaflets appeared coated with a dense fibrillar layer. The experiments suggest that, after enzyme digestion, the cytoplasmic membrane retains a coating which is rigid in the prospheroplast but which loses rigidity when the cell is transformed into a spheroplast.     

Macromolecule synthesis in yeast spheroplasts

Conditions have been established for the preparation of spheroplasts of Saccharomyces cerevisiae which are able to increase their net content of protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), several-fold upon incubation in a medium stabilized with 1 m sorbitol. The rate of RNA and protein synthesis in the spheroplasts is nearly the same as that occurring in whole cells incubated under the same conditions; DNA synthesis occurs at about half the whole cell rate. The spheroplasts synthesize transfer RNA and ribosomal RNA. The newly synthesized ribosomal RNA is incorporated into ribosomes and polysomes. The polysomes are the site of protein synthesis in these spheroplasts. Greater than 90% of the total RNA can be solubilized by treatment of the spheroplasts with sodium dodecyl sulfate or sodium deoxycholate. These spheroplast preparations appear to be a useful subject for the study of RNA metabolism in yeast.     

Reconstitution of SEC gene product-dependent intercompartmental protein transport

Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTP gamma S. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors.     

Catabolite inactivation of phosphoenolpyruvate carboxykinase in spheroplasts from Saccharomyces Cerevisiae

Catabolite inactivation of phosphoenolpyruvate carboxykinase was studied in yeast spheroplasts using 0.9 M mannitol or 0.6 M potassium chloride as the osmotic support. In the presence of potassium chloride the rate of catabolite inactivation was nearly the same as that occurring in intact yeast cells under different conditions of incubation. However, in the presence of mannitol, catabolite inactivation in spheroplasts was prevented. The mannitol inhibition of catabolite inactivation was released by addition of ammonium or phosphate ions. At a concentration of 0.3 M ammonium or 0.06 M phosphate ions, the maximum rate of catabolite inactivation in spheroplasts suspended in mannitol was achieved and was comparable with that observed in spheroplasts incubated in 0.6 M potassium chloride as the osmotic stabilizer. Sodium sulfate (0.04 and 0.4 M) or potassium chloride (0.06 and 0.6 M) did not release the mannitol inhibition of catabolite inactivation in spheroplasts. In intact yeast cells, 0.9 M mannitol, 0.08 M ammonium or 0.1 M phosphate ions did not influence the rate of catabolite inactivation. The nature of the effects of mannitol, ammonium and phosphate ions on catabolite inactivation in yeast spheroplasts is disscussed.     

Different binding sites for glucose and sorbose at the erythrocyte membrane,studied by gel filtration and infrared spectroscopy

Summary Human red blood cell membranes were solubilized with sodium dodecylsulfate and incubated with various concentrations of¹⁴C-glucose and¹⁴C-sorbose. After gel filtration on Sephadex G-100, which separated lipoproteins of differing lipid content, it was observed that the radioactivity of the bound glucose coincided with the protein peak. Radioactivity of bound sorbose was found mainly before and after the protein peak. This distribution of bound sugars was confirmed by double labeling experiments in which³H-glucose and¹⁴C-sorbose were applied simultaneously. Infrared spectroscopy revealed differences between the membranes loaded with sorbose and glucose. Particularly, the band in the C–O–C and P=O region at 1,225 cm^–1 was intensified in the sorbose-loaded membranes. Compared to serum albumin, the erythrocyte membranes were found to bind 4 times as much¹⁴C-glucose per mg of protein. It is concluded from the results obtained by gel filtration that glucose and sorbose preferentially bind at different sites of the erythrocyte membrane. The results obtained by infrared spectroscopy correspond with this conclusion.     

Characterization of the fusion of enveloped viruses with the plasma membrane of Saccharomyces cerevisiae spheroplasts

Vesicular stomatitis virus (VSV) was associated at low pH with Saccharomyces cerevisiae spheroplasts. In the cold, the association was characterized as reversible binding to the spheroplast surface. At 37 degrees C, the association became irreversible due to fusion of the viral envelope with the yeast plasma membrane according to the following data. Proteinase K digestion degraded the viral envelope glycoprotein G but left the internal N and M proteins of VSV intact and associated with the spheroplasts. The plasma membrane could be stained by indirect immunofluorescent labeling using antiserum against VSV. By immunoelectron microscopy, no VSV particles could be detected at the spheroplast surface. Instead, the G protein could be visualized at the external aspect of the plasma membrane using specific antiserum and protein A-gold. Fusion of VSV with spheroplasts occurred below pH 4.75 at temperatures of 30-42 degrees C. It was strictly dependent on the prior removal of the yeast cell wall. The fusion process was fast, calcium-independent, and nonleaky, leaving the spheroplasts viable for at least 4 h. On the average, less than 100 VSV particles could be fused per one spheroplast. Similar data were obtained with Semliki Forest virus.     

Spheroplasts of Torulopsis candida yeasts and their oxidizing activity]

The paper describes the conditions in which the spheroplasts of the yeast Torulopsis candida IBFM-Y-127 with a high respiration rate can be isolated. The preliminary incubation of the cells with SH-reagents has to be carried out in a buffer without an osmotic stabilizer, and the incubation in a medium containing 0.6 M KCL, 0.1 M MgSO4, 0.1 MKH2PO4, pH 5.2. In these conditions the cells are incubated with the enzyme from Helix pomatia during 15 to 20 minutes, and the yield of the spheroplasts is 95 to 100 per cent. The spheroplasts oxidize various substrates (glucose, acetate, ethanol) at the same, or even higher, rate as the intact cells.     

Lipid-protein interactions at the erythrocyte membrane. Different influence of glucose and sorbose on membrane lipid transition

When observed over a temperature range, erythrocyte membrane lipids undergo a transition at 18–20 °C (Zimmer, G. and Shirmer, H. (1974) Biochim. Biophys. Acta 345, 314–320). This observation has prompted an investigation of the effects that substrate binding has on the transition of the red cell membrane. Glucose and sorbose were compared, since transport kinetics of these sugars still pose unresolved questions.In membranes, preloaded with glucose, the break at the transition temperature was intensified, while it was abolished or reversed in membranes preloaded with sorbose.These results were corroborated using different solubilization procedures (sonication, sodium dodecyl sulfate treatment) of the membranes, and also different techniques (viscosimetry, 90° light scattering, 1-anilino-naphthalene-8-sulfonate fluorescence).In extracted membrane lipids, viscosimetry indicated a break at transition temperature after preloading with either glucose or sorbose.Disc electrophoresis revealed a different binding pattern of the two sugars.It is suggested, that the amplification of the discontinuity in red cell membranes by glucose and the abolition or reversal of the break by sorbose are mediated by membrane protein- and/or membrane lipid-protein interaction.     

Effect of Yeast Extract Concentration on Viability and Cell Distortion in Rhizobium spp.

A low concentration of yeast extract (0·1%) in liquid media favoured rapid growth and high percentage of viable cells in cultures of Rhizobium japonicum (CB 1809), R. lupini (WU 425), R. meliloti (SU 47), R. trifolii (TA1) and a cowpea strain (CB 756). Concentrations of yeast extract > 0·35% depressed viability and produced distorted cells in all strains except SU 47: TA1 was especially sensitive. When used at 0·5–1% (w/v), each yeast extract (Difco, Oxoid, Vegemite) or casein hydrolysate produced greatly enlarged abnormal cells of TA1, each containing several granules of poly-β-hydroxybutyrate and whorls of intracytoplasmic membranes, and showing greater internal disorganisation than that seen in root nodule bacteroids. Lysogenic and non-lysogenic cultures of R. trifolii were all sensitive to yeast extract, and such sensitivity, for strains of several species, was unrelated to effectiveness in nodulating host plants. Glycine inhibited growth of all strains tested. Several other amino acids occurring in casein hydrolysate inhibited TA1 strongly and induced formation of distorted cells and spheroplasts; this distortion was partly counteracted by adding salts of calcium or magnesium. In media with 0·1% yeast extract the use of mannitol, sucrose, lactose or galactose as alternative carbon sources, each at a concentration of 0·02–1%, did not affect numbers of viable rhizobia or cell shape in all strains tested.     

Catabolite inactivation of phosphoenolpyruvate carboxykinase in spheroplasts from Saccharomyces cerevisiae.

Catabolite inactivation of phosphoenolpyruvate carboxykinase was studied in yeast spheroplasts using 0.9 M mannitol or 0.6 M potassium chloride as the osmotic support. In the presence of potassium chloride the rate of catabolite inactivation was nearly the same as that occurring in intact yeast cells under different conditions of incubation. However, in the presence of mannitol, catabolite inactivation in spheroplasts was prevented. The mannitol inhibition of catabolite inactivation was released by addition of ammonium or phosphate ions. At a concentration of 0.3 M ammonium or 0.06 M phosphate ions, the maximum rate of catabolite inactivation in spheroplasts suspended in mannitol was achieved and was comparable with that observed in spheroplasts incubated in 0.6 M potassium chloride as the osmotic stabilizer. Sodium sulfate (0.04 and 0.4 M) or potassium chloride (0.06 and 0.6 M) did not release the mannitol inhibition of catabolite inactivation in spheroplasts. In intact yeast cells, 0.9 M mannitol, 0.08 M ammonium or 0.1 M phosphate ions did not influence the rate of catabolite inactivation. The nature of the effect of mannitol, ammonium and phosphate ions on catabolite inactivation in yeast spheroplasts is discussed.