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R Sentandreu

Publications and source records attributed to R Sentandreu.

108 records · Page 6Linked to original sources
PubMed1977

Invertase messenger ribonucleic acid in Saccharomyces cerevisiae. Kinetics of formation and decay.

Saccharomyces cerevisiae -136ts (Hutchison, H.T., Hartwell, L.H. and McLaughlin, C.S. (1969) J. Bacteriol. 99, 807-814) incubated in the presence of maltose at 23 degrees C (permissive temperature) synthesized the RNA messengers which codify derepressed invertase (an external mannoprotein) and induced alpha-glucosidase (a non-glycosylated internal enzyme). The enzymes were not synthesized if the mutant was transferred to the maltose-containing medium at the moment of incubation at 37 degrees C indicating that the cells had no pools of the specific RNA messengers and that transcription of the DNA was a prerequisite to enzyme synthesis. Cycloheximide inhibited syntheses of the enzymes both at 37 and at 23 degrees C suggesting that the enzymic activities were the result of "de novo" synthesis of the proteins and did not result from the activation of proenzymes. In derepressed cells the number of invertase mRNA molecules is probably larger than that actually being translated. The half-life of the derepressed invertase mRNA was calculated from the moment that the molecules of RNA messenger were limiting the enzyme synthesis and a value of 30-35 min was estimated. The value found for the basal (repression independent) invertase mRNA was of 45-50 min. The half-life of alpha-glucosidase mRNA was computed following the mathematical procedure described in the Appendix, and a value of 23 min was obtained. These results are consistent with the existence of relatively long-lived RNA messengers involved in the synthesis of extracellular macromolecules.

Cycloheximide↗
PubMed1977

The mechanism of catabolite inhibition of invertase by glucose in Saccharomyces cerevisiae.

Saccharomyces cerevisiae -136ts synthesized invertase in media containing maltose and sucrose. In the presence of glucose synthesis of enzyme took place when the sugar concentration was lower than 1%. At higher concentrations enzyme formation was repressed. Analysis of the glucose effect before RNA inhibition showed that the hexose interfered with the transcription of DNA into invertase messenger RNA. Translation of invertase messenger already formed was also inhibited and the kinetics of this effect was similar to that produced by cycloheximide. Invertase activity was independent of glucose suggesting that the hexose produces no catabolite inhibition of invertase activity. Inhibition of invertase translation by glucose turned out to be reversible but the amount of enzyme produced was dependent on duration of treatment. It is suggested that the catabolite repression of invertase synthesis produced by glucose operates at the levels of transcription and translation and produces an increase in the rate of mRNA degradation. The catabolite repression has no effect on secretion and does not interfere with the catalytic activity of invertase.

Cycloheximide↗
PubMed1977

Biosynthesis of the yeast cell wall: selective assays and regulation of some mannosyl transferase activities.

Assays have been developed for some transfer reactions involved in the synthesis of Saccharomyces cerevisiae wall mannoproteins, both in a particulate preparation in the presence of EDTA or Triton X-100, and after lipid extraction with chloroform-methanol at -20 C. The mannosyl transferase activities were also studied in cells made permeable to GDP-mannose by toluene-ethanol treatment ("in situ"). In these permeabilized cells, the glycosylating reactions dependent on lipid carriers (dolichol derivatives) did not function, but those independent of them were unaffected. The lipid-independent mannosyl transferase activities were partially inhibited by nucleotide diphosphates probably in a competitive manner. Increase of the nucleotide diphosphate pool "in vivo" might slow down the speed of the transfer reactions carried out by the mannan synthetase system.

Adenosine Diphosphate↗
PubMed1976

Cell wall synthesis regulation in Saccharomyces cerevisiae. Effect of RNA and protein inhibition.

In this investigation the regulation of wall formation in Saccharomyces cerevisiae ts-136 (Hutchison, H.T., Hartwell, L.H. and McLaughlin, C.S. (1969) J. Bacteriol. 99, 807-814) was analyzed by following the inhibition of RNA and protein synthesis. Lomofungin, thiolutin and 8-hydroxyquinoline at the concentrations needed to inhibit RNA synthesis also produced inhibition of glucan and mannan synthetases. The synthesis of RNA was also blocked in S. cerevisiae ts-136 by incubation at the non-permissive temperature (37 degrees C). Mannan formation decreased steadily but glucan synthesis remained after 4 to 5 h. After a few minutes of blocking protein synthesis with cycloheximide mannan synthesis was also blocked whereas glucan formation was unaffected by the presence of the drug. These results suggest a high degree of stability for glucan synthetases. S. cerevisiae ts-136 after 2 h of incubation at the non-permissive temperature (37 degrees C) showed a preferential formation of wall materials (mannan and glucan) indicating that the RNA messengers which codify wall mannan peptides have a slower decay rate than those of the cytoplasmic proteins. The data presented indicate that the existence of stable glucan synthetases and RNA messengers of the wall mannan peptides of slow decay rate results in the continuous synthesis of glucans and mannoproteins of the yeast wall throughout the cell cycle.

Anti-Bacterial Agents↗
PubMed1975

Synthesis of yeast wall glucan.

Saccharomyces cerevisiae was treated with a mixture of toluene and ethanol to make it permeable to small molecules. This treatment unmasked a glucan synthetase activity which was assayed with UDP-[U-14C]glucose. About 60% of the polymer formed was beta-(I leads to 3)glucan. No labelled lipids were detected. The 14C incorporated was recovered in a particulate membrane preparation isolated by differential centrifugation. When the particles themselves were assayed for glucosyl transfer activity none was found. The toluene-treated preparations also catalysed the transfer of mannosyl residues from GDP-mannose to polymeric materials by a process independent of glucosyl transfer.

Carbon Radioisotopes↗
PubMed1975

Site of initial glycosylation of mannoproteins from Saccharomyces cerevisiae.

The cellular site of initial glycosylation of proteins from Saccharomyces cerevisiae has been studied. Short pulses of [U-14C]mannose label the ribosomal fraction of the yeast. Most of the label was associated with polysomes; monosomes contained only a small amount of radioactivity. All of the radioactivity present in the polysomal fraction was accounted by mannose and smaller amounts of glucose and glucosamine. Puromycin treatment detached more than 50% of the radioactivity from the polysomes; treatment of polysomes at pH 10.0 also caused the release of radioactivity. These results indicate that initial sugar binding occurs while the nascent polypeptide chains are still growing on the ribosomes. When the cells were preincubated with 2-deoxy-D-glucose, incorporation of [U-14C]mannose into the polysomes and the cell wall was inhibited, whereas its incorporation into membrane fractions was unimpaired. It was concluded that 2-deoxy-D-glucose inhibited the synthesis of glycoproteins by interference with the initial glycosylation steps at the ribosomal level.

Carbon Radioisotopes↗
PubMed1969

Yeast cell-wall synthesis.

1. A study of wall synthesis has been made by following the incorporation of radioactive glucose and threonine into the cytoplasm and wall of yeast. 2. Both glucose and threonine are incorporated into a mannan glycopeptide. The glucose is also synthesized into a structural glucan of the wall. 3. The mannan glycopeptide contains high-molecular-weight mannan and low-molecular-weight mannose and oligosaccharide units composed of mannose. Both types of carbohydrate are attached to the peptide. The extent of radioactive incorporation into these different carbohydrate constituents of the glycopeptide remained constant during a pulse-chase experiment. No evidence of a sequential synthesis of oligosaccharides and high-molecular-weight mannan was obtained. 4. Cycloheximide inhibits the incorporation of threonine into the wall but only partially inhibits the incorporation of glucose. Thus not all the polysaccharide deposited into the wall is dependent on a simultaneous peptide synthesis and incorporation. 5. Protoplasts grown in an iso-osmotic medium secreted a mannan polymer that was probably a glycopeptide.

Carbohydrates↗
PubMed1968

The structure of a glycopeptide isolated from the yeast cell wall.

1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a beta-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the beta-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the beta-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate-amino acid linkages found in the glycoprotein.

Aspartic Acid↗
PubMedDate not supplied

Electrophoretic mobility of external invertase from free and gel-immobilized yeast cells.

Electrophoretic mobility of secreted invertase (E.C. 3.2.1.26) from gelatin-immobilized yeast cells was analysed and compared with that of secreted invertase from freely suspended batch-grown cells. Invertase from immobilized cells showed a lower mobility after 24 h of incubation, in medium containing either glucose or raffinose as carbon source. Changes in invertase mobility were also followed in a time course both for immobilized and for freely suspended batch-grown cells. Mobility of invertase from free cells increased after approximately 15 h of incubation, independently of the carbon source, whilst that of invertase from immobilized cells remained constant. The differences observed were attributed to a different level of glycosylation of the protein moiety in free and immobilized cells. The amount of mannoproteins in the cell walls of immobilized cells was also investigated by ConA-ferritin labelling and quantification of ferritin particle density in ultrathin sections; the results of this experiment showed a higher content of mannoproteins in the walls of immobilized cells when compared with free cells. As a whole, these results are indicative of physiological changes that can be ascribed to the peculiar microenvironment of gel-immobilized cells.

Blotting, Western↗
PubMedDate not supplied

Initial steps of wall protoplast regeneration in Candida albicans.

Cell wall regeneration of individual Candida albicans yeast and mycelial protoplasts was studied with confocal and electron microscopy using polyclonal antibodies and lectins. Quantitative measurements of the fluorescence emitted by individual protoplasts during the process of regeneration indicate that chitin is the first polymer to be laid down, whereas beta (1,3)- and beta (1,6)glucan are incorporated at a later stage. Mannoproteins were found on the surface of fresh protoplasts and those newly synthesized were then deposited with time. During the first steps of wall regeneration, the proteins that interacted covalently with chitin or glucan were different, but the same species were found linked to each polymer in yeast and mycelial regenerating forms. The aggregates formed by regenerating protoplasts were shown to be due to the chitin and mannoprotein network initially laid.

Blotting, Western↗
PubMedDate not supplied

Cloning of cDNAs coding for Candida albicans cell surface proteins.

Two cDNA libraries were constructed from mRNAs obtained from yeast cells and germ-tubes of Candida albicans in lambda gt11. Immunoscreening with polyclonal antibodies raised against cell wall components allowed the detection of 29 positive clones. Two of these clones were selected for their specific reactivity with antisera either from yeast (clone 11Y) or germ-tubes (clone 24M). cDNA fragments were isolated by the digestion of lambda DNA with EcoRI. Southern blot analysis with these fragments as probes demonstrated homology with C. albicans DNA, and by Northern analysis two mRNAs transcripts were detected with sizes of approximately 1.5 kb for 11Y and 1.1 kb for 24M. Both transcripts were present in yeast cells as well as in germ-tubes. The whole genes were isolated from a C. albicans genomic library in the YRp7 vector by hybridization with the cDNA probes. Monospecific antibodies were purified from polyclonal antisera by affinity for the fusion proteins. Western blot analysis with 11Y-specific antibodies revealed a cross-reactivity with material found in the yeast cell wall as well as in other subcellular fractions, whereas clone 24M codes for a 30 kDa protein detected mainly in the membrane fraction and in the SDS-solubilized material from mycelial cell walls. Sequencing of the cDNA molecules and restriction map of the cloned genes demonstrate that clone 11Y is an enolase previously characterized in C. albicans, whereas clone 24M does not show significant homology with any other cloned gene.

Amino Acid Sequence↗