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E Cabib

Publications and source records attributed to E Cabib.

At least 37 records · Page 2Linked to original sources
PubMed1991

Proteinase B is, indeed, not required for chitin synthetase 1 function in Saccharomyces cerevisiae.

Previous genetic evidence led to the conclusion that proteinase B of yeast was not involved in the function of chitin synthetase 1 (Chs1), based on the demonstration of normal septum formation, cell division and chitin deposition in mutants devoid of the proteinase (Zubenko, G.S., Mitchell, A.P., and Jones, E.W. (1979) Proc. Natl. Acad. Sci. USA 76, 2395-2399). Later, however, it was found that the essential enzyme for septum formation is chitin synthetase 2, whereas Chs1 acts as an auxiliary enzyme, whose absence results in daughter cell lysis under acidic conditions (Cabib, E., Sburlati, A., Bowers, B. and Silverman, S.J. (1989) J. Cell Biol. 108, 1665-1672). By using the lytic behavior as a criterion, we have now found that prb1 strains are not defective in Chs1 function. Certain strains contain a recessive suppressor of lysis which could mask the Chs1 defect. However, appropriate crosses and transformation experiments showed that the prb1 mutants do not harbor the suppressor. It may now be concluded with confidence that proteinase B is not required for chitin synthetase 1 function.

Base Sequence↗
PubMed1991

CAL1, a gene required for activity of chitin synthase 3 in Saccharomyces cerevisiae.

The CAL1 gene was cloned by complementation of the defect in Calcofluor-resistant calR1 mutants of Saccharomyces cerevisiae. Transformation of the mutants with a plasmid carrying the appropriate insert restored Calcofluor sensitivity, wild-type chitin levels and normal spore maturation. Southern blots using the DNA fragment as a probe showed hybridization to a single locus. Allelic tests indicated that the cloned gene corresponded to the calR1 locus. The DNA insert contains a single open-reading frame encoding a protein of 1,099 amino acids with a molecular mass of 124 kD. The predicted amino acid sequence shows several regions of homology with those of chitin synthases 1 and 2 from S. cerevisiae and chitin synthase 1 from Candida albicans. calR1 mutants have been found to be defective in chitin synthase 3, a trypsin-independent synthase. Transformation of the mutants with a plasmid carrying CAL1 restored chitin synthase 3 activity; however, overexpression of the enzyme was not achieved even with a high copy number plasmid. Since Calcofluor-resistance mutations different from calR1 also result in reduced levels of chitin synthase 3, it is postulated that the products of some of these CAL genes may be limiting for expression of the enzymatic activity. Disruption of the CAL1 gene was not lethal, indicating that chitin synthase 3 is not an essential enzyme for S. cerevisiae.

Amino Acid Sequence↗
PubMed1991

The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle.

The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.

Cell Division↗
PubMed1991

Differential inhibition of chitin synthetases 1 and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycins.

Polyoxin D, nikkomycin X, and nikkomycin Z are all competitive inhibitors of chitin synthetase 2 (Chs2), the essential enzyme for primary septum formation in Saccharomyces cerevisiae, and of Chs1, a repair enzyme. However, Chs2 is more resistant to these antibiotics than Chs1. When Co2+, the best stimulator of Chs2, was used in the assay for this enzyme, the differences in the Ki values for nikkomycins between the two isozymes reached 3 orders of magnitude. These results point to differences in the active sites of the two isozymes. Polyoxin D was much more effective than nikkomycin Z in inhibiting cell growth. This underlines the importance of the choice of enzyme and of assay conditions when cell wall-synthesizing enzymes are used in screens for possible antifungal agents.

Aminoglycosides↗
PubMed1989

Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae.

Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.

Acetylglucosamine↗
PubMed1988

Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae.

Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomyces cerevisiae, is not required for synthesis of the chitinous primary septum. The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for the newly found chitin synthase 2, a relatively minor activity in yeast. Disruption of the chitin synthase 2 gene results in the loss of well-defined septa and in growth arrest, establishing that the gene product is essential for both septum formation and cell division.

Cell Division↗
PubMed1988

Fungal cell wall synthesis: the construction of a biological structure.

The cell wall is of vital importance for the protection of the fungal cell. It consists of structural components, mostly beta-linked polysaccharides, and of interstitial components, usually glycoproteins. Cell shape is determined by the wall and is attained by localized growth. Regulatory mechanisms that act on chitin and beta(1----3) glucan synthetase indicate how localized biosynthesis can be achieved. Whereas structural polysaccharides appear to be exported by vectorial synthesis through the plasma membrane, glycoproteins are manufactured in an assembly line that takes them through several cellular compartments before exocytosis to the periplasmic space. In this space, the last phase of cell wall construction takes place, i.e. branching of structural polysaccharides and linkage between different components.

Cell Wall↗
PubMed1986

Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae.

Strains containing a disrupted structural gene for chitin synthetase (chs1::URA3) are defective in chitin synthetase 1 (Chs1) activity but contain normal amounts of chitin (Bulawa, C.E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, L., and Robbins, P. W. (1986) Cell 46, 213-225). We have now detected in such strains a new chitin synthetase activity (Chs2), at levels about 5% of those of Chs1 in wild-type cells. Thus, Chs2 is presumably the physiological agent for chitin deposition in strains with a disrupted CHS1 gene and probably also in wild-type strains. Chs1 and Chs2 share certain properties, such as stimulation by N-acetylglucosamine and by partial proteolysis. They differ sharply, however, in divalent cation specificity and in pH optimum. Chs2 also shows less sensitivity than Chs1 to inhibition by polyoxin D or sodium chloride, a property that was used to demonstrate the presence of Chs2 in wild-type extracts. As in the case of Chs1, most of the Chs2 activity was found to be associated with the plasma membranes. This finding, together with the apparent zymogenic nature of Chs2, is consistent with the hypothesis, previously put forward for Chs1, that localized deposition of chitin is attained by activation of the zymogen form at a specific time and place. Function and significance of the two chitin synthetases are discussed in connection with fungal morphogenesis and evolution.

Cations, Divalent↗
PubMed1986

The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo.

The chitin synthase of Saccharomyces is a plasma membrane-bound zymogen. Following proteolytic activation, the enzyme synthesizes insoluble chitin that has chain length and other physical properties similar to chitin found in bud scars. We isolated mutants lacking chitin synthase activity (chs1) and used these to clone CHS1. The gene has an open reading frame of 3400 bases and encodes a protein of 130 kd. The fission yeast S. pombe lacks chitin synthase and chitin. When a plasmid encoding a CHS1-lacZ fusion protein is introduced into S. pombe, both enzymatic activities are expressed in the same ratio as in S. cerevisiae, demonstrating that CHS1 encodes the structural gene of chitin synthase. Three CHS1 gene disruption experiments were performed. In all cases, strains with the disrupted gene have a recognizable phenotype, lack measurable chitin synthase activity in vitro but are viable, contain normal levels of chitin in vivo, and mate and sporulate efficiently.

Amino Acid Sequence↗
PubMed1986

The effect of papulacandin B on (1----3)-beta-D-glucan synthetases. A possible relationship between inhibition and enzyme conformation.

The antibiotic, papulacandin B, inhibited growth or (1----3)-beta-D-glucan synthetase (or both) in the fungi Saccharomyces cerevisiae, Hansenula anomala, Neurospora crassa, Cryptococcus laurentii, Schizophyllum commune and Wangiella dermatitidis. No effect was observed on Achlya ambisexualis. There was no apparent correlation between the inhibition of growth and that of the synthetase. With most of the fungal extracts, the inhibition of glucan synthetase by papulacandin B became less pronounced as the substrate (UDP-glucose) concentration was decreased. At very low levels of UDP-glucose, with the enzymes from S. cerevisiae and W. dermatitidis, the antibiotic stimulated the activity of glucan synthetase. As further studied with the W. dermatitidis enzyme, those low concentrations of UDP-glucose corresponded to a sigmoidal portion of the rate vs. substrate curve. The sigmoid segment of the curve extended to higher concentrations of UDP-glucose as the temperature was increased. Concomitantly, the range of substrate concentrations at which papulacandin B stimulated the reaction or was noninhibitory was broadened. It is tentatively concluded that glucan synthetase may exist in more than one interconvertible form. The stimulatory effect of papulacandin B is possibly due to preferential binding to the active form of the enzyme. The equilibrium between these forms could be shifted by structural changes in the membrane in which the enzyme is embedded. The lack of correlation between the effects of papulacandin B in whole cells and in extracts is discussed in terms of the variations in membrane structure in the two situations.

Aminoglycosides↗
PubMed1986

Regulation of fungal cell wall growth: a guanine nucleotide-binding, proteinaceous component required for activity of (1----3)-beta-D-glucan synthase.

By treatment with detergent and NaCl, particulate (1----3)-beta-D-glucan synthase (EC 2.4.1.34) from Hansenula anomala or Neurospora crassa was dissociated into a "soluble fraction" and a "membrane fraction." Each fraction alone was almost inactive, but enzymatic activity could be reconstituted by mixing the two fractions and adding GTP or one of its analogs. Based on their lability to heat and to incubation with trypsin, the activity in both fractions is proteinaceous. The active component in the soluble fraction appears to bind guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), since it was specifically protected by this nucleotide against heat inactivation and against inactivation in the presence of EDTA. Furthermore, precipitation of the soluble component with ammonium sulfate in the presence of GTP[gamma S] gave rise to a fraction that was highly active in the absence of added nucleotide, indicating either tight binding or covalent interaction between GTP[gamma S] and the soluble component. The membrane fraction probably contains the catalytic moiety, because it was partially protected against heat inactivation by the substrate, UDP-glucose. Soluble fractions that stimulated membrane fractions from H. anomala and N. crassa were obtained from several other fungi, including Saccharomyces cerevisiae. We propose that the soluble fraction contains a GTP-binding protein that modulates the biosynthesis of (1----3)-beta-D-glucan of fungal cell walls and probably has a major role in the regulation of cell wall morphogenesis.

Cell Membrane↗
PubMed1985

Modification of yeast plasma membrane density by concanavalin A attachment. Application to study of chitin synthetase distribution.

Yeast protoplasts were coated with different amounts of concanavalin A. Upon subsequent lysis and centrifugation in isopycnic density gradients, it was found that the buoyant density of plasma membranes was progressively increased from 1.125 to 1.21, according to the amount of bound concanavalin A. Enzymes that are attached to the plasma membrane showed the same density modifications and could thus be distinguished from constituents of intracellular membranes and organelles. With this methodology, it was confirmed that about two-thirds of yeast chitin synthetase is associated with the plasma membrane. The remainder of the enzyme was found in a peak at a lower density. Vanadate-sensitive ATPase showed a similar pattern, whereas GDP-mannose dolichyl-phosphate mannosyltransferase, an enzyme attached to the endoplasmic reticulum, remained in the same position in the gradients, irrespective of the amount of concanavalin A associated with the plasma membrane. Potential applications of this technique to the determination of plasma membrane markers and to the separation of subcellular organelles are discussed.

Adenosine Triphosphatases↗
PubMed1985

Stimulation of beta(1----3)glucan synthetase of various fungi by nucleoside triphosphates: generalized regulatory mechanism for cell wall biosynthesis.

Particulate fractions from the taxonomically diverse fungi Achlya ambisexualis, Hansenula anomala, Neurospora crassa, Cryptococcus laurentii, Schizophyllum commune, and Wangiella dermatitidis were found to catalyze the time-dependent incorporation of glucose from UDP-[14C]glucose into a water-insoluble material. The reaction was stimulated by bovine serum albumin. The product was characterized as beta(1----3)glucan on the basis of its resistance to alpha- and beta-amylase and susceptibility to beta(1----3)glucanase. With the exception of the preparation from A. ambisexualis, all others were stimulated by nucleoside triphosphates and their analogs. The best activators were GTP and guanosine 5'-(gamma-thio)triphosphate. It is concluded that the stimulation by nucleotides, previously found with the glucan synthetase of Saccharomyces cerevisiae, is a regulatory mechanism that was well conserved during fungal evolution, presumably because of its importance in controlling cell wall biosynthesis and cell growth.

Cell Wall↗
PubMed1985

Formation of septum-like structures at locations remote from the budding sites in cytokinesis-defective mutants of Saccharomyces cerevisiae.

Cell wall structures that partition membrane-bound portions of cytoplasm were formed at sites along the peripheral wall when a cytokinesis-defective cell division cycle mutant (cdc3) of Saccharomyces cerevisiae was grown at a restrictive temperature. The appearance of these structures, as observed in electron micrographs, was similar to that of normal septa. Aberrant septa were also detected in cytokinesis mutants harboring mutations cdc10, cdc11, and cdc12, after growth at 37 degrees C. Formation of the abnormal septa was abolished by the introduction, in a cdc3-containing strain, of additional cell cycle mutations that precluded events leading to cytokinesis and cell division. These results showed that septum formation can occur in the absence of cytokinesis. Formation of the abnormal structures was controlled by the same sequences of cell cycle events as formation of normal septa but was not subject to the spatial controls that ensure association of the septum with the budding site.

Cell Division↗
PubMed1984

Isolation of chitin synthetase from Saccharomyces cerevisiae. Purification of an enzyme by entrapment in the reaction product.

Chitin synthetase, in the zymogen form, was extracted with digitonin from a particulate fraction from Saccharomyces cerevisiae and converted into active form by treatment with immobilized trypsin. When the activated enzyme was incubated with UDP-GlcNAc and other components of an assay mixture, a chitin precipitate formed, trapping a large portion of the synthetase. The enzyme was easily extracted frm the chitin gel with a recovery of approximately 50% and an enrichment of approximately 100-fold. Further purification was obtained by repeating the chitin step. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the purified synthetase showed a major band corresponding to Mr 63,000, a weaker band at Mr 74,000, and some other minor bands. Under nondenaturing conditions, an Mr of 570,000 was calculated for the enzyme from Stokes radius and sedimentation coefficient determinations. After electrophoresis in a nondenaturing gel and incubation with the components of the standard assay, chitin was formed and precipitated in the gel, yielding an opaque band. Soluble oligosaccharides were not precursors for insoluble chitin, suggesting that synthesis of chitin chains takes place by a processive mechanism. N-Acetylglucosamine stimulated the purified synthetase only slightly and did not participate as a primer in the reaction. The same chain length, somewhat more than 100 units of GlcNAc, was determined in samples of chitin that had been synthesized either in vivo, or with a membrane preparation or with purified synthetase. These results suggest that chitin synthetase itself is capable both of initiating chitin chains without a primer and of determining their length.

Cell Membrane↗