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F M Klis

Publications and source records attributed to F M Klis.

At least 55 records · Page 3Linked to original sources
PubMed1995

Covalent association of beta-1,3-glucan with beta-1,6-glucosylated mannoproteins in cell walls of Candida albicans.

Yeast and hyphal walls of Candida albicans were extracted with sodium dodecyl sulfate (SDS). Some of the extracted proteins reacted with a specific beta-1,6-glucan antiserum but not with a beta-1,3-glucan antiserum. They lost their beta-1,6-glucan epitope after treatment with ice-cold aqueous hydrofluoric acid, suggesting that beta-1,6-glucan was linked to the protein through a phosphodiester bridge. When yeast and hyphal walls extracted with SDS were subsequently extracted with a pure beta-1,3-glucanase, several mannoproteins that were recognized by both the beta-1,6-glucan antiserum and the beta-1,3-glucan antiserum were released. Both epitopes were sensitive to aqueous hydrofluoric acid treatment, suggesting that beta-1,3-glucan and beta-1,6-glucan are linked to proteins by phosphodiester linkages. The possible role of beta-glucans in the retention of cell wall proteins is discussed.

Antibody Specificity↗
PubMed1994

Glucomannoproteins in the cell wall of Saccharomyces cerevisiae contain a novel type of carbohydrate side chain.

Mannoproteins in the walls of mnn9 cells of Saccharomyces cerevisiae were released by laminarinase, and purified by concanavalin A affinity chromatography and ion-exchange chromatography. Carbohydrate analysis revealed that they contained N-acetylglucosamine, mannose, and glucose. An antiserum raised against beta (1-6)-glucan reacted with four proteins with molecular masses of 66, 100, 155, and 220 kDa, respectively. Recognition by the antiserum was competitively inhibited by beta (1-6)-glucan, but not by beta (1-3)-glucan, mannan, or dextran (an alpha (1-6)-glucan). Mild periodate treatment of the wall proteins completely abolished recognition by the antiserum. Glucose-containing side chains were isolated and compared with N- and O-carbohydrate side chains. The glucose-containing side chains consisted of about equal amounts of glucose and mannose and some N-acetylglucosamine, and were larger than N-chains. They were, however, not extended N-chains, because after acetolysis, which preferentially cleaves (1-6)-linkages, their elution profiles differed strongly. A model is presented of how glucose-containing side chains might anchor mannoproteins into the glucan layer of the cell wall.

Acetylglucosamine↗
PubMed1994

Glucosylation of chimeric proteins in the cell wall of Saccharomyces cerevisiae.

Extension of a reporter protein with the carboxyterminal thirty amino acids of the cell wall mannoprotein alpha-agglutinin of Saccharomyces cerevisiae resulted in incorporation of the chimeric protein in the cell wall. By Western analysis it was shown that the incorporated protein contained beta-1,6-glucan similar to endogenous cell wall proteins, whereas excreted reporter protein was not glucosylated. This suggests that beta-1,6-glucan is involved in anchoring mannoproteins in the cell wall.

Cell Wall↗
PubMed1994

A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white.

To study cell wall assembly, a simple screening method was devised for isolating cell wall mutants. Mutagenized cells were screened for hypersensitivity to Calcofluor White, which interferes with cell wall assembly. The rationale is that Calcofluor White amplifies the effect of cell wall mutations. As a result, the cells stop growing at lower concentrations of Calcofluor White than cells with normal cell wall. In this way, 63 Calcofluor White-hypersensitive (cwh), monogenic mutants were obtained, ordered into 53 complementation groups. The mannose/glucose ratios of the mutant cell walls varied from 0.15 to 3.95, while wild-type cell walls contained about equal amounts of mannose and glucose. This indicates that both low-mannose and low-glucose cell wall mutants had been obtained. Further characterization showed the presence of three low-mannose cell wall mutants with a mnn9-like phenotype, affected, however, in different genes. In addition, four new killer-resistant (kre) mutants were found, which are presumably affected in the synthesis of beta 1,6-glucan. Most low-glucose cell wall mutants were not killer resistant, indicating that they might be defective in the synthesis of beta 1,3-glucan. Eleven cwh mutants were found to be hypersensitive to papulacandin B, which is known to interfere with beta 1,3-glucan synthesis, and four cwh mutants were temperature-sensitive and lysed at the restrictive temperature. Finally, nine cwh mutants were hypersensitive to caffeine, suggesting that these were affected in signal transduction related to cell wall assembly.

Aminoglycosides↗
PubMed1994

The linkage of (1-3)-beta-glucan to chitin during cell wall assembly in Saccharomyces cerevisiae.

Pulse-chase experiments with [14C]glucose demonstrated that in the cell wall of wild-type Saccharomyces cerevisiae alkali-soluble (1-3)-beta-glucan serves as a precursor for alkali-insoluble (1-3)-beta-glucan. The following observations support the notion that the insolubilization of the glucan is caused by linkage to chitin: (i) degradation of chitin by chitinase completely dissolved the glucan, and (ii) disruption of the gene for chitin synthase 3 prevented the formation of alkali-insoluble glucan. These cells, unable to form a glucan-chitin complex, were highly vulnerable to hypo-osmotic shock indicating that the linkage of the two polymers significantly contributes to the mechanical strength of the cell wall. Conversion of alkali-soluble glucan into alkali-insoluble glucan occurred both early and late during budding and also in the ts-mutant cdc24-1 in the absence of bud formation.

Cell Wall↗
PubMed1994

Domain conservation in several volvocalean cell wall proteins.

Based on our previous work demonstrating that (SerPro)x epitopes are common to extensin-like cell wall proteins in Chlamydomonas' reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)10 oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugametos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)x-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from Volvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.

Algal Proteins↗
PubMed1994

Identification of beta-1,6-glucosylated cell wall proteins in yeast and hyphal forms of Candida albicans.

Several cell wall proteins released from yeast and hyphal cells of Candida albicans by laminarinase reacted with an affinity-purified antiserum raised against beta-1,6-glucan. Binding of the antiserum was competitively inhibited by beta-1,6-glucan, but not by beta-1,3-glucan or isolated N-chains. Immunodetection was completely abolished when the proteins were treated with periodate. These results demonstrate that the laminarinase-released wall proteins of C. albicans possess an epitope consisting of beta-1,6-linked glucose residues. The yeast form of C. albicans contained four beta-1,6-glucosylated wall proteins, an Endo H-resistant protein of 125 kDa and three glycoproteins which became only detectable after Endo H digestion and had a molecular mass of 320, 170 and 44 kDa, respectively. As for the hyphal form, a different set of beta-1,6-glucosylated wall proteins was found consisting of two Endo H-resistant glycoproteins of 125 and 80 kDa, respectively, and two glycoproteins that after Endo H digestion had a molecular mass of 320 and 38 kDa, respectively. Sodium dodecyl sulfate-extractable wall proteins and medium proteins did not react with the beta-1,6-glucan antiserum. The beta-1,6-glucan epitope could be removed by aqueous hydrofluoric acid indicating that the epitope is phosphodiester-linked to the cell wall proteins. It is speculated that the epitope forms part of a GPI-anchor and might be involved in the anchoring of mannoproteins into the cell wall.

Candida albicans↗
PubMed1993

Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae.

The sexual adhesion protein of Saccharomyces cerevisiae MAT alpha cells, alpha-agglutinin, could not be extracted from the cell wall with hot sodium dodecyl sulfate (SDS), but became soluble after digestion of the cell wall with laminarinase. This indicates that it is intimately associated with cell wall glucan. A fusion protein was constructed consisting of the signal sequence of yeast invertase, guar alpha-galactosidase, and the C-terminal half of the alpha-agglutinin. Most of the fusion protein was incorporated in the cell wall. A small amount could be extracted with SDS, but most of it could only be extracted with laminarinase. On the other hand, cells containing a construct consisting of the signal sequence of invertase and alpha-galactosidase released most of the alpha-galactosidase into the medium and all cell wall-associated alpha-galactosidase was released by SDS. Labelling with antibodies showed that the alpha-galactosidase part of the fusion protein was exposed on the surface of the cell wall. The results demonstrate that the C-terminal half of the alpha-agglutinin contains the information needed to incorporate a protein into the cell wall.

Biological Transport↗
PubMed1991

Cell wall glucomannoproteins of Saccharomyces cerevisiae mnn9.

Mannoproteins were isolated from Saccharomyces cerevisiae mnn9 mutant cell walls by laminarinase digestion and purified by affinity and anion-exchange chromatography. The purified mannoprotein fraction contained three predominant proteins with molecular masses of 300 kDa, 220 kDa and 160 kDa. These compounds were absent in an SDS extract of cell walls or in a hot-citrate extract of mnn9 cells. The carbohydrate part of the purified mannoproteins consisted of (N-acetyl)glucosamine, mannose and glucose in a molar ratio of 1:53:4. O-Glycosidically linked chains, containing 70% of the mannose, were released by mild beta-elimination. N-Glycosidically linked chains, representing 80% of the (N-acetyl)glucosamine and 20% of the mannose, were released by peptide N-glycosidase F (PNGase F) digestion. Complete degradation of protein by alkaline hydrolysis released besides the N- and O-glycosidically linked chains, another type of carbohydrate chain containing the residual (N-acetyl)glucosamine, mannose and most of the glucose in a molar ratio of 1:17:18. Glucose was beta-glycosidically linked. The results indicate that beta-glucose is linked to PNGase F-resistant N-linked chains present on cell wall mannoproteins. We propose that these chains are responsible for the linkage between mannoproteins and glucan in the cell wall.

Acetylglucosamine↗
PubMed1987

Isolation and composition of the constitutive agglutinins from haploid Saccharomyces cerevisiae cells.

Sex-specific agglutinins from the cell surface of haploid cells of Saccharomyces cerevisiae (X2180, mta and mt alpha) were purified and analysed. The constitutive agglutinin from mta cells was extractable with 3 mM dithiothreitol. It was shown to be a glycoprotein (3% mannose) with an apparent Mr of 43,000 based on gel filtration, but in SDS-PAGE it behaved as a much smaller molecule (Mr between 18,000 and 26,000). About one in three amino acids was a hydroxyamino acid. Its biological activity was resistant to boiling for 1 h, but sensitive to pronase. Intact mt alpha cells retained their agglutinability in the presence of dithiothreitol but limited trypsinizing released a biologically active agglutinin fragment. It had an apparent Mr of 320,000 (gel filtration). When analysed by SDS-PAGE, a single diffuse band with an apparent Mr of 225,000 was observed. The protein was 94% (w/w) mannose with a trace of N-acetyl glucosamine. Its biological activity was almost completely lost after boiling for 1 h. Both agglutinins behaved as monovalent molecules and specifically inhibited the biological activity of both noninduced and pheromone-induced cells. Pheromone treatment of mta cells resulted in an apparent 32-fold increase in agglutinin activity at the cell surface, whereas pheromone treatment of mt alpha cells only doubled the apparent agglutinin activity.

Agglutinins↗
PubMed1985

Sexual Agglutination in the Unicellular Green Alga Chlamydomonas eugametos: Identification and Properties of the Mating Type plus Agglutination Factor.

Gametes of the unicellular green alga Chlamydomonas eugametos agglutinate via their flagella. The mating type plus agglutination factor was solubilized by relatively mild treatments such as a short pH shock or an osmotic shock indicating that it is an extrinsic membrane component. It was also extracted in the nonionic detergent Triton X-100. A simple two-step procedure consisting of gel filtration over Sepharose 4B-cross-linked followed by anion exchange chromatography of the void volume yielded an electrophoretically pure preparation of a single high molecular weight glycoprotein. The agglutination factor sedimented as a 9.3 S particle (assuming a density of 1.50) in sucrose gradients. This low value, compared with the high apparent molecular weight seen during gel filtration and electrophoresis, suggests that the agglutination factor is a rod-like molecule. This was confirmed by viewing rotary-shadowed preparations in the electron microscope. A population of long slender molecules was revealed (328 +/- 20 nanometers), many of which had a knob at one end and a flexible region about one fourth of the length from the other end.

Journal Article↗
PubMed1981

Arabinogalactan Protein from a Crude Cell Organelle Fraction of Phaseolus vulgaris L.

Sonication of a crude cell organelle fraction from hypocotyl tissue of dark-grown bean seedlings, and from suspension-cultured cells released a hydroxyproline-containing protein. The purification of this protein is described. It was found to be an arabinogalactan protein composed of 90% carbohydrate and 10% protein. The major sugars are galactose, arabinose, and uronic acids, and the major amino acids are hydroxyproline, serine, and alanine. Its molecular weight was estimated at 1.4 x 10(5) daltons and the isoelectric point at pH 2.3. The molecule is soluble in 5% trichloroacetic acid and can be precipitated with beta-galactosyl Yariv antigen. Pulse-chase experiments indicated that it was a secretory protein. The biosynthesis of arabinogalactan proteins is discussed.

Journal Article↗
PubMed1981

Hydroxyproline Glycosides in Secretory Arabinogalactan-Protein of Phaseolus vulgaris L.

The carbohydrate moiety of secretory arabinogalactan protein in bean seedlings (Phaseolus vulgaris L. cv. Prélude) is attached to the peptide backbone through hydroxyproline, serine, and threonine. Hydroxyproline-linked side chains, consisting of arabinose, galactose, glucose, and rhamnose, comprise the major part of the sugar residues. These hydroxyproline glycosides differ from those in non-extractable cell wall protein but show similarities with those in wall protein of the alga Chlamydomonas.

Journal Article↗
PubMed1976

Glycosylated seryl residues in wall protein of elongating pea stems.

The protein content of salt-washed cell walls isolated from etiolated stems of Pisum sativum L. approximately doubled during elongation. In the same period the concentration in the wall of hydroxyproline, hydrazine-labile (= presumably glycosylated) serine, valine, tyrosine, lysine, and histidine increased markedly in comparison with other amino acids. After elongation was completed both the amino acid composition and the protein content of the cell wall changed only slightly. The ratio for the wall of hydrazine-labile seryl residues to hydroxyprolyl residues remained constant during and after elongation and was found to be 0.20. A linear relationship was established between the rate of elongation and the concentration in the wall of the hydroxyproline-rich glycoprotein both in vivo and in cut sections incubated in buffer.

Journal Article↗
PubMedDate not supplied

An assay of relative cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe.

We have developed a new assay to determine relative cell wall porosity in yeasts, which is based on polycation-induced leakage of UV-absorbing compounds. Polycations with a small hydrodynamic radius as measured by gel filtration (poly-L-lysine) caused cell leakage independent of cell wall porosity whereas polycations with a large hydrodynamic radius (DEAE-dextrans) caused only limited cell leakage due to limited passage through the cell wall. This allowed the ratio between DEAE-dextran- and poly-L-lysine-induced cell leakage to be used as a measure of cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe. Using this assay, we found that the composition of the growth medium affected cell wall porosity in S. cerevisiae. In addition, we could show that cell wall porosity is limited by the number of disulphide bridges in the wall and is dependent on cell turgor. It is argued that earlier methods to estimate cell wall porosity in S. cerevisiae resulted in large underestimations.

Cell Wall↗
PubMedDate not supplied

The glucanase-soluble mannoproteins limit cell wall porosity in Saccharomyces cerevisiae.

The cell wall porosity of batch-grown Saccharomyces cerevisiae was maximal in the early exponential phase and fell off rapidly to lower levels in later growth phases. Treatment of stationary-phase cells with alpha-mannosidase restored wall porosity to the level of cells in early exponential phase. When cells in the early exponential phase were treated with alpha-mannosidase, or tunicamycin, an inhibitor of N-glycosylation, even higher porosities were obtained. Mutants with truncated mannan side-chains in their wall proteins also had very porous walls. The importance of the mannan side-chains for wall porosity was also seen during sexual induction. Treatment with alpha pheromone, which leads to the formation of wall proteins with shorter mannan side-chains, enhanced wall porosity. Disulphide bridges also affect cell wall porosity. They were predominantly found in the glucanase-soluble wall proteins. Because the main part of the mannan side-chains is also found in this family of wall proteins, our results demonstrate that the glucanase-soluble mannoproteins limit cell wall porosity in yeast.

Benzenesulfonates↗
PubMedDate not supplied

Cyclic variations in the permeability of the cell wall of Saccharomyces cerevisiae.

To study cell-cycle-related variations in wall permeability of Saccharomyces cerevisiae, two approaches were used. First, an asynchronous culture was fractionated by centrifugal elutriation into subpopulations containing cells of increasing size. The subpopulations represented different stages of the cell cycle as judged by light microscopy. Cell wall porosity increased when these subpopulations became enriched with budded cells. Secondly, synchronous cultures were obtained by releasing MATa cells from alpha-factor induced G1-arrest. These cultures grew synchronously for at least two generations. The cell wall porosity increased sharply in these cultures, shortly before buds became visible and was maximal during the initial stages of bud growth. It decreased in cells which had completed nuclear migration and before abscission of the bud had occurred. The porosity reached its lowest value during abscission and in unbudded cells. We examined the incorporation of mannoproteins into the wall during the cell cycle. SDS-extractable mannoproteins were incorporated continuously. However, the incorporation of glucanase-extractable mannoproteins, which are known to affect cell wall porosity, showed cyclic oscillations and reached its maximum after nuclear migration. This coincided with a rapid decrease in cell wall porosity, indicating that glucanase-extractable mannoproteins might contribute to this decrease.

Benzenesulfonates↗