Biogenesis of the yeast cell wall.
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Biological Transport↗
Biomedical subjects
R Sentandreu
Publications and source records attributed to R Sentandreu.
Localization of Chitin synthase in Mucor rouxii by an autoradiographic method.
The localization of chitin synthase in the cells of Mucor rouxii was studied by a method which combined permeabilization of the cells with toluene/ethanol and incubation with the radioactive substrate UDP-[3H]GlcNAc followed by high resolution autoradiography. By this technique it was demonstrated that most of the chitin synthesized by these cells was located within the cytoplasm, and only a small amount of the enzyme product appeared at the cell surface. It was concluded that most of the chitin synthase of M. rouxii is located in the cytoplasm of the cells.
Autoradiography↗
Synthesis and assembly of wall polymers on regenerating yeast protoplasts.
Accumulation of chitin and glucan on S. cerevisiae and C. albicans protoplasts begins shortly after resuspension in the regeneration medium, and mannoprotein molecules also appear retained by the regenerating wall after 30-60 minutes in S. cerevisiae or after a longer lag period in C. albicans. Nevertheless, a considerable fraction of the synthesized mannoproteins, which in SDS-acrylamide gels exhibit a different pattern from that of wall manno-proteins of cells, are still released to the growth medium during at least eight hours. De novo synthesis of chitin synthase, but not of glucan synthase, is observed in S. cerevisiae from about 30 minutes after initiation of the regeneration process. The interaction between microfibrils of nascent chitin formed by C. albicans protoplasts is altered by strains as Calcofluor White or Congo Red. In the presence of the former one, no microcrystalline lattice of the polymer is formed and protoplasts do not regenerate correctly.
Candida albicans↗
Cell wall composition and protoplast regeneration in Candida albicans.
The transition of blastospores to the mycelial phase in Candida albicans was induced after the blastospores were kept at 4 degrees C for several hours and then transferred to a fresh medium prewarmed at 37 degrees C. Glucan was the most abundant polymer in the wall in the two morphogenetic forms but the amount of chitin was higher in the mycelial form than in blastospores. Efficient protoplasting required reducing agents and proteases together with beta-glucanases (zymolyase). Protein synthesis in regenerating protoplasts was initiated after about 30 min. Chitin synthetase, initially very low, was incorporated in important amounts into cell membranes mainly in a zymogenic state. After a few hours chitin was the most abundant polymer found in the aberrant wall of the regenerating protoplast.
Candida albicans↗
Calcofluor white alters the assembly of chitin fibrils in Saccharomyces cerevisiae and Candida albicans cells.
In the presence of calcofluor white, budding scars and dividing cross-walls of Saccharomyces cerevisiae exhibited fluorescence, indicating that the brightener was a specific marker of fungal chitin. In addition, incubation of cells in the presence of the brightener did not stop protein and wall-polymer formation, but abnormal deposition of chitin occurred. Chitin synthesis was normal in regenerating protoplasts of Candida albicans in the presence of calcofluor, but formation of the crystalline lattice was blocked. These results suggest that crystallization of nascent subunits may occur by a self-assembly mechanism that was blocked by the stain.
Benzenesulfonates↗
Turnover of protein components of the plasma membrane of Saccharomyces cerevisiae.
The peptide composition of plasma membrane in Saccharomyces cerevisiae cells growing at different temperatures between 18 and 38 degrees C was studied using SDS-polyacrylamide gel electrophoresis. Stability of the proteins, both qualitative and quantitative, was observed at the tested temperatures. Treatment for 2 h with cycloheximide decreased by about 50% the amount of a 80 kDa membrane peptide at 18, 23, 28 and 33 degrees C, with no other apparent effects. At 38 degrees C the 80 kDa peptide was not affected by the presence of the drug. Addition of tunicamycin to cultures at concentrations partially inhibitory to growth caused a large accumulation of the 80 kDa peptide in the plasma membrane, which cycloheximide did not modify. Pulse-chase experiments indicated a low rate of turnover of total plasma membranes in cells growing at 18 and 28 degrees C. In contrast, at 38 degrees C about 50% of the radioactivity in plasma membranes disappeared after a 2 h chase. The 80 kDa protein band was the only one with significant differential decay.
Cell Membrane↗
Regulation of alpha-galactosidase synthesis in Saccharomyces cerevisiae and effect of cerulenin on the secretion of this enzyme.
Saccharomyces cerevisiae -136ts MEL10 (a thermosensitive mutant whose RNA synthesis is inhibited at 37 degrees C but is normal at 23 degrees C), when grown at 23 degrees C in the presence of galactose, melibiose or L-arabinose, these cells synthesize alpha-galactosidase mRNA. In the simultaneous presence of both galactose and glucose the transcription of alpha-galactosidase mRNA is blocked. Glucose also interferes with mRNA translation, but the degree of inhibition depends on concentration and time of addition of the hexose to induced cells. It has been found that the final concentration of alpha-galactosidase produced by induced cells when transferred at the non-permissive temperature (37 degrees C) is inversely proportional to the incubation time in glucose. Cerulenin inhibits lipid formation on growing yeasts, but protein synthesis and selective permeability are not affected. The antibiotic partially inhibits secretion of alpha-galactosidase with a parallel accumulation of this enzyme in membranous structures, specially at the level of the plasma membrane. Induction of alpha-galactosidase or cerulenin addition to growing cells, results in changes in the polypeptide composition of the plasma membrane.
Antifungal Agents↗
The plasma membrane of Saccharomyces cerevisiae. Molecular structure and asymmetry.
The molecular structure of the plasma membrane of the haploid strain Saccharomyces cerevisiae X-2180 1 A has been studied by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein and glycoprotein components have been identified and their apparent Mr determined. A glycoprotein showing an apparent Mr of 27500 has been shown to be the main structural component. Treatment of the cells with cycloheximide prior to plasma membrane isolation resulted in a redistribution of the relative amounts of each protein band and a drastic reduction in the number of Schiff positive bands. It is postulated that treatment with this drug rids the plasma membrane of glycoprotein secretory components which are in the process of being secreted to the periplasmic space, thus allowing the study of the basic structural components of the organelle. The electrophoretic pattern of the internal membranes revealed close similarities with that of the plasma membrane and though two-dimensional electrophoresis might disclose greater differences, these similarities suggest a common origin for most of the components of both membranous systems. Finally, radioiodination techniques, have been used in studying the asymmetric disposition of some of the components of the plasma membrane. At least five polypeptides were identified as located to the outer layer of the plasma membrane and two more glycopeptides were shown to span across the bilayer.
Cell Membrane↗
Mutants of Saccharomyces cerevisiae cell division cycle defective in cytokinesis. Biosynthesis of the cell wall and morphology.
The four temperature-sensitive mutants of Saccharomyces cerevisiae in the cell division cycle defective in cytokinesis (cdc, 3, 10, 11 and 12), have been analyzed with respect to the biosynthesis of the cell wall polymers. After 3 hours of incubation at the non-permissive temperature (37 degrees C) these strains stop growing. The synthesis of glucan, mannan and chitin (wall polymers) level off in a similar time, but glucan, mannan and chitin synthases remained active for at least 4 hours. If the mutants are analyzed by transmission and scanning electron microscopy different pictures emerge. Two of the mutants cdc 10 and cdc 12, after 3 hours of incubation at 37 degrees C present apparently normal cytoplasms and cell wall surfaces with multiple elongated buds. The other two mutants, cdc 3 and cdc 11, present a completely disarranged cytoplasmic content and damage at the level of the plasma membrane is evident. These and other observations, suggest that between the execution points of cdc 3 (0.27) and cdc 10 (0.58), essential processes in the assembly of cell membrane occur.
Cell Division↗
Metabolism of Saccharomyces cerevisiae envelope mannoproteins.
By pulse and chase labeling experiments, two independent mannoprotein pools have been found associated with the Saccharomyces cerevisiae envelope. One of them probably corresponds to mannoproteins localized in the periplasmic space. These molecules showed a high turnover rate at 28 degrees C. The second pool is formed by intrinsic wall mannoproteins which are apparently stable for long periods of time, after a small initial turnover. These results suggest that at least part of the mannoproteins initially found in the periplasmic space may move into the wall. The time lag between the addition of the radioactive precursors and their incorporation in the cell envelope (20-30 min for amino acids and about 10 min for carbohydrate) indicates that protein formation and carbohydrate incorporation take place in succession. Moreover, bulk glycosylation of mannoproteins seems to occur close in time to the moment of secretion into the periplasmic space.
Cell Wall↗
Uptake of sucrose by Saccharomyces cerevisiae.
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Antimycin A↗
Regulation of invertase synthesis by glucose in Saccharomyces cerevisiae.
Saccharomyces cerevisiae growing under repressible conditions (1% of glucose or more) produces a burst of external invertase when shifted to higher temperatures. The secretion of this invertase requires protein synthesis, but was found to be independent of RNA formation. The level of mRNA accumulated and translated was inversely proportional to the glucose present in the growth medium. These results are consistent with the hypothesis that invertase is continuously synthesized both in the presence and absence of glucose, but under repressible conditions is degraded before secretion takes place.
Enzyme Repression↗
Metabolic relationship between invertase and acid phosphatase isoenzymes in Saccharomyces cerevisiae.
Repressed cells of Saccharomyces cerevisiae, subjected to inhibition of both RNA and protein synthesis, showed a pattern of membrane-bound and cytosol acid phosphatase to the external enzyme which seemed to be linked through a precursor-product relationship. Gel exclusion chromatography did not indicate clear differences between the isoenzymes. Moreover, centrifugation experiments in CsCl and precipitation with concanavalin A suggested that there were no acid phosphatase molecules devoid of carbohydrate. Membrane-bound invertase displayed a molecular weight and a carbohydrate to protein ratio smaller than those of the exocellular enzyme. The values of molecular weight and buoyant density of the membrane-bound enzyme were closer to those found for the cytosol invertase. The stability of the level of the soluble invertase detected in the cytoplasm under derepression conditions, or after RNA or protein synthesis inhibition was found to be only apparent and represented the result of an equilibrium between synthesis and degradation.
Acid Phosphatase↗
Regulation of acid phosphatase synthesis in Saccharomyces cerevisiae.
In Saccharomyces cerevisiae-136ts (Hutchison, H.T., Hartwell, L.H. and McLaughlin, C.S. (1969) J. Bacteriol. 99, 807--814) derepressed acid phosphatase was almost exclusively located outside the permeability barrier. Only a minor part of the activity was associated with the protoplasts; about half of it (48%) in the soluble fraction, the rest bound to the internal (45%) and plasma (7%) membranes. The activity found in the membranes of derepressed cells decreased by 30--40% after addition of inorganic phosphate or cycloheximide suggesting that this activity is the precursor of the external enzyme. The alkaline phosphatase activity level could not be modified by changes in the concentration of inorganic phosphate. Acid phosphatase was not synthesized if the cells were transferred to a low phosphate medium at the moment of incubation at 37 degrees C or in the presence of cycloheximide at 23 degrees C. The data suggested that enzyme formation is the result of the transcription and translation of a specific gene(s) and not the activation of a proenzyme. Inorganic phosphate did not inhibit the translation of mRNA though it may act at the level of the transcription.
Acid Phosphatase↗
The plasma membrane of Saccharomyces cerevisiae. Isolation and some properties.
The isolation of Saccharomyces cerevisiae plasma membrane was carried out after hypotonic lysis of yeast protoplasts treated with concanavalin A by two independent methods: a, at low speed centrifugation and b, at high speed centrifugation in a density gradient. Several techniques (electron microscopic, enzymic, tagging, etc.) were used to ascertain the degree of purification of the plasma membranes obtained. The low speed centrifugation technique as compared with the other method gave a higher yield of plasma membranes with a similar degree of purification. Analysis of the yeast plasma membrane of normally growing cells by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed at least 25 polypeptide bands. Twelve glycoprotein bands were also found, and their apparent molecular weights were determined. Treatment of the protoplasts with cycloheximide resulted in a significant decrease in the carbohydrate and protein content of the plasma membrane. The electrophoretic pattern of the plasma membrane of cycloheximide-treated cells showed a redistribution of the relative amounts of each protein band and a drastic reduction in the number of Schiff-positive bands. The isoelectric point of the most abundant proteins was low (pI 4) or lower than expected from previous data. A large part of the mannosyl transferase activity found in the cell (80%) was associated with the internal membranes, the remaining activity (20%) was located in the plasma membrane preparation. Part of the mannosyl transferase activity of the cells is located at the plasma membrane surface. Invertase (an external mannoprotein) is found in both the plasma and internal membranes, and as the specific activity dropped significantly following cycloheximide treatment of the cells, it is suggested that these membranes systems are the structures for the glycosylation of a precursor invertase and its subsequent release into the periplasmic space. Other transferase found in the plasma membrane preparation transfers glucose residues from UDPglucose to a poly(alpha(1 leads to 4) polymer identified as glycogen.
Carbohydrates↗
Inositol deficiency in yeast: metabolic, enzymatic and autoradiographic studies.
The addition of inositol to starved cells of Saccharomyces cerevisiae NCYC 86 resulted in an initiation of growth. Inositol was incorporated into phosphatidyl-inositol and after a lag period RNA was the first macromolecule with a rate of synthesis departing from the rate observed in deprived cells. Pulse chase experiments showed that inositol was first incorporated into phosphatidylinositol and later into more polar lipids. Finally it appeared to be excreted into the surrounding medium. When S. cerevisiae NCYC 86 was grown in suboptimal concentrations of inositol (0,5 microgram/ml), alterations in the level of some membrane-bound enzymatic activities were detected; these might reflect structural modifications of the cellular membranes due to a different composition of phospholipids. High-resolution autoradiography showed that inositol was probably first incorporated into internal membranes and later transferred to the plasma membrane. Analytical experiments carried out with inositol-deprived cells showed that inositol was released into the surrounding medium in that case. The unbalanced growth detected in S. cerevisiae NCYC 86 under inositol deprivation might be due to an abnormal functioning of the cell membranes as a consequence of the deficiency in inositol-containing phospholipids.
Autoradiography↗
Inositol deficiency in Saccharomyces cerevisiae NCYC 86.
When Saccharomyces cerevisiae NCYC 86, an inositol dependent strain, is grown at suboptimal concentrations of inositol, the buds are apparently unable to separate from the parent cells. Thin sections of such cells show an irregularly thickened cell wall. These morphological features may be due to a continuation or increase in the production of glucan while the synthesis of DNA, RNA, phospholipids.and protein is greatly inhibited.
Cell Wall↗
Biosynthesis of Saccharomyces cerevisiae glycoproteins: nature of some participating glycolipids.
A particulate membrane preparation from Saccharomyces cerevisiae catalyzed the incorporation of mannose from GDP-mannose into lipids that were extractable in chloroform-methanol. One lipid has been previously characterized as dolichyl phosphomannose. Another one was purified by chromatography on silicic acid, DEAE-cellulose and Sephadex LH-20 was found to be alkali unstable. The lipid moiety was shown to be dolichol and the glycosydic part contained mannose, glucose and glucosamine. Radioactive mannose was also incorporated at a slower rate into more polar compounds. They were soluble in chloroform-methanol-water and were seen to liberate neutral oligosaccharides after alkaline hydrolysis. Radioactive mannose was also incorporated into substances which behave chemically as glycoproteins since they were insoluble in organic solvents, water and trichloroactic acid. Pronase treatment of the trichloroacetic acid-insoluble material released water-soluble oligosaccharides. When the particulate preparation which had been extracted with chloroform-methanol at-20 C, was incubated with GDP-(U-14C)mannose, radioactivity was incorporated into glycolipids that were soluble in chloroform-methanol-water and into glycoproteins. This result suggests that at least part of the mannose was transferred to endogenous acceptors independent of dolichyl phosphomannose.
Cell-Free System↗