Osmotic imbalance in inositol-starved spheroplasts of Saccharomyces cerevisiae.

Physiological states associated with inositol starvation of spheroplasts of Saccharomyces cerevisiae were investigated and compared with conditions preceding death of starved whole cells. In the absence of synthesis of inositol-containing lipids, cell surface expansion terminated after one doubling of whole cells. In spheroplasts, cessation of membrane expansion was apparently followed by rapid development of an osmotic imbalance, causing lysis. Continued synthesis and accumulation of cytoplasmic constituents within the limited cell volume were implicated as a cause of the osmotic imbalance. In whole cells, an increase in internal osmotic pressure also follows termination of membrane and cell wall expansion. The cell wall prevents lysis, allowing a state of increasing cytoplasmic osmotic pressure to persist in the period preceding onset of inositol-less death.     

Extensive MHC class I-restricted CD8 T lymphocyte responses against various yeast genera in humans

The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood. While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts. The yeast-specific CD8 T cells expressed interferon-gamma but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells. Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall. Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections. And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses.     

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.     

A simple assay for optimizing yeast-mammalian cell fusion conditions

Polyethylene glycol (PEG)-induced cell fusion can be a useful method for the transfer of yeast artificial chromosomes (YACs) from yeast spheroplasts to mammalian cells in culture, although success varies between recipient cell types. Experiments aimed at determining optimum fusion conditions can also be very time-consuming. To minimize this difficulty, a reporter plasmid has been constructed that allows yeast-mammalian cell fusion rates to be determined within 3 d. The speed and sensitivity of the assay should allow a more systematic evaluation of cell lines for their capacity to fuse with yeast, and for rapid optimization of fusion parameters.     

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.     

Copper reduction by yeast cell wall materials and its role on copper uptake in Debaryomyces hansenii

Copper binding reducing activities of cell wall materials (CWM) prepared from cells of the yeast Debaryomyces hamsenii were examined. When CWM was treated with copper sulfate (0.1 mM CuSO₄), the copper was partially reduced from Cu (II) to Cu (I) and bound to CWM (below 10 nmol per mg dry wt.). The bound copper was mostly in the fraction of mannan-protein. Both copper-binding ability and protein content decreased with protease treatments. Mannan-protein prepared from CWM bound more copper than mannan did. This suggests that Cu (II) bound to the protein portion in CWM and was reduced to Cu (I). The optimum pH of copper reduction by CWM was about 5.0. The amount of copper bound to CWM increased with reducing agents and decreased with oxidizing agents. On the other hand, the copper uptake by yeast whole cells and spheroplasts was also stimulated by reducing agents, but inhibited by oxidizing agents. Furthermore, copper uptake by spheroplasts was stimulated in the presence of CWM. The optimum pH of copper uptake coincided with that of copper reducing activity. These results suggest that yeast cell wall not only supplies copper binding but also reduces copper, and the reduced copper is transported into yeast cells. The yeast cells may have copper-reducing proteins in the cell wall.     

Uptake and distribution of chromium in Saccharomyces cerevisae exposed to Cr(III)-organic compounds

Abstract

The present study investigates the influence of different Cr(III)-organic compounds [Cr(III)-citrate and Cr(III)-histidine] in growth-nonsupportive exposure medium on the uptake and localisation of chromium in the cell structure of the yeast Saccharomyces cerevisae. The amount of total accumulated chromium in yeast cells and the distribution of chromium between the yeast cell walls and spheroplasts were determined by atomic absorption spectroscopy. Chromium accumulation potential was shown to depend on treatment time, metal concentration as well as the nature of the bound ligand. Chromium uptake was characterised by a time-dependent increase of total chromium which suggests that the amount of cell-accumulated chromium also tended to increase over time. Cellular chromium accumulation (mg g^?1 dry wt) of Cr(III)-histidine is higher than Cr(III)-citrate. The pH dependence pattern of chromium accumulation is similar for both of the Cr(III)-organic compounds: pH 6.5>pH 5>pH 8. Substantial differences were found between the two Cr(III)-organic compounds, in the total chromium accumulation as well as in the distribution in yeast cell walls and spheroplasts.     

A simple method for culture and stable maintenance of giant spheroplasts from the yeast Saccharomyces cerevisiae

When spheroplasts of the yeast Saccharomyces cerevisiae are cultured in liquid medium containing osmotic stabilizer, they undergo nuclear division and growth without cell division, resulting in the formation of giant spheroplasts with multinuclei. In this study, we report a simple method for the culture and stable maintenance of giant spheroplasts. The selection of culture media and cell concentration was found to be important for the growth and maintenance of giant spheroplasts. Among the conditions that we tested, static culture in a synthetic Burkholder's medium in 96-well U-bottomed culture plates was most effective. Under appropriate conditions, we could maintain giant spheroplasts for more than 6 days without proliferation of whole cells or marked lysis. The average diameter of spheroplasts can vary from 16 to 53μm, depending on their initial concentration.     

Controlled production of spheroplasts in the yeast Nadsonia elongata

Summary Yeast cells of Nadsonia elongata were cultivated in such a way that simultaneously with enzymatic lysis of the cell wall a partial synthesis of cell wall components was taking place. After the initial period of cultivation, which lasted about 10 h and during which the morphology of cells remained unchanged when compared to controls, the cells were transformed into prospheroplasts. The prospheroplasts were larger than the control cells and, though they enlarged in volume in distilled water, they still retained the shape of the original cells. However, some changes were found in the ultrastructure of the cell walls of prospheroplasts in comparison with that of the cell walls of intact cells: while in yeast cells the surface was smooth, in prospheroplasts the fibrillar network was revealed as a result of the removal of the amorphous component; the gradual disappearance of the outer cell wall layer and a swelling of the remaining cell wall fragment were seen in ultrathin sections. After about 20-h cultivation the prospheroplasts were transformed into spheroplasts. The spheroplasts were osmotically fragile, and did not retain the shape of the yeast cell, even in isoosmotic environment. On the surface of spheroplasts only the fibrillar network composed of separate fibrils was seen. The spheroplasts were the final stage of yeast cell transformation under the conditions employed in the present study. Under the mentioned conditions true protoplasts are never formed. However, if the synthesis of cell wall components could not take place simultaneously with the lysis of the cell wall, the cells were transformed to protoplasts.     

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.     

Reversion of protoplasts and spheroplasts in the yeast Nadsonia elongata

Summary The time rate of regeneration of the cell wall and reversion of protoplasts of the yeast Nadsonia elongata to cells of normal shape and size has been compared with the capability for regeneration of spheroplasts of this yeast. Nearly all protoplasts in a given culture were able to regenerate new walls and had usually reverted to cells of normal appearance by the 30th h of cultivation. Spheroplasts required only half this time to do this. These results can be interpreted as evidence that regeneration of a wall by protoplasts does not depend upon the presence of a cell wall primer, because the proportion of reverting protoplasts (which lack wall remnants) was the same as that of reverting spheroplasts (which possess them). The presence of wall remnants in spheroplasts appears to have merely an accelerating effect on the formation of a new wall and on subsequent reversion of the spheroplasts to complete cells of normal shape and size.     

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.     

An efficient technique for the isolation of yeast spores and the preparation of spheroplast lysates active in protein synthesis

A procedure for isolation of yeast spores and preparation of yeast spheroplasts with the use of the bacterial lytic enzyme, Zymolyase, is described. The high lytic activity of Zymolyase, allows isolation of the yeast spores in a rapid and simple manner. The resulting spores are not contaminated with vegetative cells and retain their full activity in germination. Moreover, the enzyme appears to be very efficient in preparation of yeast lysates, actively synthesizing proteins. The use of Zymolyase for other purposes is suggested.     

In vivo functional assay of a recombinant aquaporin in Pichia pastoris

The water channel protein PvTIP3;1 (alpha-TIP) is a member of the major intrinsic protein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-type P. pastoris displayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts of P. pastoris expressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of the Escherichia coli glycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.     

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.     

Cell cycle inhibition of yeast spheroplasts

Summary Osmotically stabilized yeast spheroplasts are capable of extensive DNA synthesis. Although the rate of DNA synthesis in spheroplasts is approximately one-third that of intact cells, the relative amounts of nuclear and mitochondrial DNA synthesized by spheroplasts is very similar to the relative amounts synthesized by intact cells. Furthermore, nuclear but not mitochondrial DNA synthesis is inhibited in MATa spheroplasts by the application of the yeast mating pheromone, -factor. Similarly, DNA synthesis is reversibly temperature-sensitive in spheroplasts created from cdc7 and cdc8 mutant cells.     

Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes.

Two Yeast Artificial Chromosomes (YACs) were isolated each with a full-length copy of the human gene that encodes the trifunctional protein containing phosphoribosylglycinamide synthetase (GARS), phosphoribosylglycinamide formyltransferase (GART) and phosphoribosylaminoimidazole synthetase (AIRS). The YACs were characterized by restriction mapping and by in situ hybridization of cosmid subclones containing the YAC ends to human metaphase chromosomes. One of the YACs contains co-cloned non-contiguous DNA whereas the other appears to have a single 600 kbp insert from 21q22.1, the location of the GART gene. A restriction map of the gene was obtained from two cosmid subclones which together span the 40 kb gene. The gene is functional when YAC DNA is transferred into GARS- or GARS-and-AIRS-deficient Chinese Hamster Ovary cells. The gene transfer was carried out both by lipofection using purified yeast DNA and by fusion between yeast spheroplasts and the hamster cells. Restriction analysis of DNA from cell lines whose purine auxotrophy was complemented by the YAC showed that with either method a complete and unrearranged copy of the gene can be transferred. The majority of the fusion cell lines appear to contain at least 80% of the YAC.     

Relationship between the replicative age and cell volume in Saccharomyces cerevisiae

Reaching the limit of cell divisions, a phenomenon referred to as replicative aging, of the yeast Saccharomyces cerevisiae involves a progressive increase in the cell volume. However, the exact relationship between the number of cell divisions accomplished (replicative age), the potential for further divisions and yeast cell volume has not been investigated thoroughly. In this study an increase of the yeast cell volume was achieved by treatment with pheromone alpha for up to 18 h. Plotting the number of cell divisions (replicative life span) of the pheromone-treated cells as a function of the cell volume attained during the treatment showed an inverse linear relationship. An analogous inverse relationship between the initial cell volume and replicative life span was found for the progeny of the pheromone-treated yeast. This phenomenon indicates that attaining an excessive volume may be a factor contributing to the limitation of cellular divisions of yeast cells.     

The human HPRT gene on a yeast artificial chromosome is functional when transferred to mouse cells by cell fusion

A 680-kb yeast artificial chromosome (YAC) that contains a functional copy of the human hypoxanthine phosphoribosyltransferase (HPRT) gene has been isolated. This YAC, yHPRT, and another YAC, yXY837, which contains the 3' end of the HPRT gene, have been mapped with restriction enzymes that cleave human DNA infrequently. The HPRT gene lies near the center of yHPRT. Fusion of yHPRT-containing yeast spheroplasts with mouse L A-9 cells, which are HPRT-negative, gives rise to HPRT-positive colonies. These colonies contain the human HPRT gene and express human HPRT mRNA. Fusion of yeast with mammalian cells is an efficient way of testing the integrity and functionality of human DNA contained in YACs.