Table 3 - uploaded by Enrico Cabib
Content may be subject to copyright.
Source publication
Digitonin treatment at 30 degrees C of a Saccharomyces cerevisiae mutant lacking proteinase B permeabilized the cells and caused rapid and extensive activation of chitin synthetase in situ. The same result was obtained with a mutant generally defective in vacuolar proteases. By lowering the temperature and using different permeabilization procedure...
Contexts in source publication
Context 1
... in chitin synthetase activation with growth phase and strain used. When cells har- vested from logarithmic-or early stationary- phase cultures were treated with digitonin in parallel experiments, the early stationary-phase cells yielded almost twice as much chitin synthe- tase activity as did those in logarithmic phase (Table 3, experiment 1). In late stationary phase, however, activation declined (data not shown). ...
Context 2
... late stationary phase, however, activation declined (data not shown). With cells from the wild-type strain the maxi- mum level of synthetase exceeded that of the protease B-deficient mutant harvested at the corresponding growth phase (Table 3). Mutant pep4-3, which is defective in several vacuolar proteases (7), showed almost as much activation as did its parental strain (Table 3). ...
Context 3
... cells from the wild-type strain the maxi- mum level of synthetase exceeded that of the protease B-deficient mutant harvested at the corresponding growth phase (Table 3). Mutant pep4-3, which is defective in several vacuolar proteases (7), showed almost as much activation as did its parental strain (Table 3). ...
Similar publications
The treatment of Candida albicans (yeast form) with digitonin or dimethyl sulfoxide permeabilized cells and caused the activation of chitin synthase in situ. Endogenous activation was completely prevented by the sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoic acid); partially prevented by the proteas...
Citations
... Secretion of recombinant proteins may be accelerated by cell permeabilization. Procedures used for permeabilization usually involve treatment of cells with organic solvents or detergents [8,9]. Other methods involve repeated freezing and thawing [10], air-drying [11], freeze-drying [12], or osmotic shock [13] of the cells. ...
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the production of granulocytes, macrophages, and white blood cells. The effects of osmotic pressure on secretion of human GM-CSF into the culture medium were investigated in suspension cultures of transgenic tobacco cells. An increase in osmotic pressure caused by the addition of mannitol decreased the cell size index, with the effect being more pronounced when cells were measured wet rather than dry. Increased osmotic pressure enhanced the secretion of hGM-CSF. At 90 g/liter mannitol, the maximum concentration tested, hGM-CSF was present in the culture medium at 980 μg/liter. As the concentration of mannitol increased, the total amount of protein secreted also increased, but was disproportionately enriched in GM-CSF. NaCl, another osmoticum, had very similar effects on cell growth and hGM-CSF production, but did not cause enrichment for hGM-CSF.
... Glucan synthase assays were performed as described by Kang and Cabib (1986). Chitin synthase 1 assays of digitonin-treated cells were performed as described (Fernandez et al., 1982). ...
Five sequences were isolated by selection for multiple copy plasmids that conferred resistance to laminarinase, an enzyme that specifically degrades cell wall beta(1-3) glucan linkages. Strains carrying three of these plasmids showed alterations in cell wall glucan labelling. One of these plasmids carried PBS2, a previously identified, non-essential gene which produces a variety of phenotypes and encodes a mitogen-activated protein kinase kinase analogue (Boguslawski and Polazzi, 1987). Cells carrying PBS2 at multiple copy show a small decrease in cell wall beta(1-6) glucans. Measurements of beta(1-3) glucan synthase activity in multi-copy PBS2 cells showed an approximate 30-45% increase in enzyme specific activity while a pbs2 delta disruption strain showed a decrease in glucan synthase activity of approximately 45% relative to control. A pbs2 delta disruption strain was laminarinase super-sensitive and supersensitive to K1 killer toxin while a strain carrying PBS2 at multiple copy was resistant to killer toxin. A second plasmid carried a portion of the MHP1 gene which has been reported to encode a microtubule-interacting protein (Irminger-Finger et al., 1996). The MHP1 gene product is a predicted 1398 amino acid protein and only approximately 80% of the amino portion of this protein is required for laminarinase resistance. Cells carrying the amino portion of MHP1 at multiple copy show a decrease in high molecular weight cell wall beta(1-6) glucans and were killer toxin resistant while a disruption strain was viable and killer toxin super-sensitive. Cells carrying this plasmid showed decreased levels of high molecular weight beta(1-6) glucans and increased glucan synthase activity. The laminarinase resistance conferred by the third plasmid mapped to the previously uncharacterized YCL051W open reading frame and this gene was therefore named LRE1 (laminarinase resistance). The LRE1 gene encodes a non-essential 604 amino acid hydrophilic protein. Unexpectedly, cells carrying LRE1 at multiple copy show no alteration in cell wall glucans or glucan synthase activity. Subcloning experiments demonstrated that the production of these cell wall effects requires the presence of both LRE1 and YCL052C (PBN1), a second open reading frame present on the original plasmid. Cells carrying multiple copies of PBN1 alone show no significant alterations in cell wall glucans or glucan synthase activity, indicating that these effects require the presence of multiple copies of both genes.
... To test for Myc-Chslp function, CSI activity was measured in digitonin-permeabilized wild-type, chsl::HIS3, and MYC-CHS1 strains (Fernandez et al., 1982). chsl:.HIS3 strains had approximately 10% of the activity of wild-type cells. ...
In Saccharomyces cerevisiae, the synthesis of chitin, a cell-wall polysaccharide, is temporally and spatially regulated with respect to the cell cycle and morphogenesis. Using immunological reagents, we found that steady-state levels of Chs1p and Chs3p, two chitin synthase enzymes, did not fluctuate during the cell cycle, indicating that they are not simply regulated by synthesis and degradation. Previous cell fractionation studies demonstrated that chitin synthase I activity (CSI) exists in a plasma membrane form and in intracellular membrane-bound particles called chitosomes. Chitosomes were proposed to act as a reservoir for regulated transport of chitin synthase enzymes to the division septum. We found that Chs1p and Chs3p resided partly in chitosomes and that this distribution was not cell cycle regulated. Pulse-chase cell fractionation experiments showed that chitosome production was blocked in an endocytosis mutant (end4-1), indicating that endocytosis is required for the formation or maintenance of chitosomes. Additionally, Ste2p, internalized by ligand-induced endocytosis, cofractionated with chitosomes, suggesting that these membrane proteins populate the same endosomal compartment. However, in contrast to Ste2p, Chs1p and Chs3p were not rapidly degraded, thus raising the possibility that the temporal and spatial regulation of chitin synthesis is mediated by the mobilization of an endosomal pool of chitin synthase enzymes.
... The fraction for determination of intracellular chitinase was the 1OOOOO g supernatant of the extract obtained by cell disruption with glass beads. was assayed on cells permeabilized with digitonin as reported by Fernandez et al. (1982). Chs2 and Chs3 are not detected with this assay if present at wild-type levels. ...
Previous results [E. Cabib, A. Sburlati, B. Bowers & S. J. Silverman (1989) Journal of Cell Biology 108, 1665-1672] strongly suggested that the lysis observed in daughter cells of Saccharomyces cerevisiae defective in chitin synthase 1 (Chs1) was caused by a chitinase that partially degrades the chitin septum in the process of cell separation. Consequently, it was proposed that in wild-type cells, Chs1 acts as a repair enzyme by replenishing chitin during cytokinesis. The chitinase requirement for lysis has been confirmed in two different ways: (a) demethylallosamidin, a more powerful chitinase inhibitor than the previously used allosamidin, is also a much better protector against lysis and (b) disruption of the chitinase gene in chs1 cells eliminates lysis. Reintroduction of a normal chitinase gene, by transformation of those cells with a suitable plasmid, restores lysis. The percentage of lysed cells in strains lacking Chs1 was not increased by elevating the chitinase level with high-copy-number plasmids carrying the hydrolase gene. Furthermore, the degree of lysis varied in different chs1 strains; lysis was abolished in chs1 mutants containing the scs1 suppressor. These results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting.
... One of each of the ensuing yeast transformants was grown in minimal medium with either glucose or galactose as the carbon source. Chitin synthase activity was measured in digitonin-treated cells (17) . All five showed approximately wild-type levels in the glucose-grown cultures whereas the galactose-grown cultures had from four to 20 times the wild-type activity (data not shown). ...
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.
... The supernatant fluid (zymogen) was used as source of enzymes. Chitin Synthase Assays For a semiquantitative measurement of chitin synthase activity, assays were performed on digitonin-permeabilized cells (Fernandez et al., 1982). Washed cells (10-100 mg wet weight) were suspended in three volumes of 1% digitonin in 25 mM 2[N-morpholinojethanesulfonic acid (MES) (pH 6.3). ...
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.
... It also compares chitin synthase in permeabilized cells and spheroplast membranes. Activation appears to be similar to that recently described in S. cerevisiae (20). ...
... The resulting membranes were used immediately. Permeabilization with digitonin, dimethyl sulfoxide (DMSO), and toluene-ethanol was done as described previously (1,20,31). Cells were washed with 10 volumes of 50 mM Tris hydrochloride (pH 7.5) containing 0.2% digitonin in the case of digitonin permeabilization, and then they were resuspended in 1 volume of the same buffer. ...
... The chitin synthase assay used for C. (lbicalns in the present study was that previously used for S. ceresisiae (20) with minor modifications. The volume of the incubation mixture was reduced to 25 [lI to conserve enzyme and substrates. ...
The treatment of Candida albicans (yeast form) with digitonin or dimethyl sulfoxide permeabilized cells and caused the activation of chitin synthase in situ. Endogenous activation was completely prevented by the sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoic acid); partially prevented by the protease inhibitors antipain, leupeptin, and N alpha-tosyl-L-lysyl chloromethyl ketone; and also partially prevented by EDTA. Thus, a clostripain-like protease may be involved in the endogenous activation phenomenon. The pH activity profile, cofactor requirements, and kinetic parameters of the endogenously activated chitin synthase were identical to those of the trypsin-activated enzyme in protoplast membranes.
... Permeabilized cell chitin synthetase assay. The procedure we used to assay for chitin synthetase activity in permeabil-ized cells is a modification to the referenced protocol (7). Briefly, cells from an overnight culture at 37°C were added to fresh medium and grown to the mid-logarithmic phase. ...
The natural polyoxins are a class of peptidyl nucleoside antibiotics produced by Streptomyces and characterized by their ability to compete with UDP-GlcNAc for active site binding to chitin synthetase (Hori, et al., 1971 and 1974). As chitin synthetase inhibitors they offer the promise of a new source for clinical antifungal agents. Previous reports have demonstrated that the polyoxins are strong inhibitors of chitin synthetase in vitro, effective at µM concentrations against chitin synthetase of C. albicans (Becker, et al., 1983). However, when assayed for toxicity to zoopathogenic fungi such as Candida albicans or Cryptococcus neoformans concentrations in the mM range were required to observe toxic effects (Becker, et al., 1983).
There are three roles for chitinases in fungi:
(a)
Most spectacularly, they are involved in the gross autolysis associated with the release of spores in some basidiomycete fruit bodies. Examples include the maturation of puffballs, Lycoperdon species1, and the autodigestion of gill tissue following spore release in the ink-caps, Coprinus species2.
(b)
They have a nutritional role. In the case of soil saprophytes such as Aspergillus species3,4 and Trichoderma species5, chitinase enables them to utilise chitinous debris from dead invertebrates and fungi as food sources. In the case of pathogens of crustacea, insects and fungi, chitinases also enable them to penetrate their hosts. Examples include the crayfish pathogen Aphanomyces astaci
6, insect pathogens such as Beauvaria and Cordyceps species7,8. There is however no convincing evidence that there is any appreciable re-cycling of the chitin in cell walls9,10 even though chitinase accumulates to a marked extent in old cultures11,12,i.e. there does not seem to be any appreciable autophagic utilisation of chitin in starving cultures
(c)
They have a morphogenetic role in the growth and differentiation of all chitin-containing fungi. This, the most fundamental of the three roles, has however proved the most difficult to obtain evidence for. Indeed some authors question whether chitinase and other lytic enzymes are involved in hyphal apical growth. Burnett13 suggests that “teleologically speaking, the apex would seem to be a most dangerous location for a lytic entity!”, and Wessels14 proposes a model for hyphal growth that does not require wall lytic enzymes. The unitary model for hyphal growth proposed by Bartnicki-Garcia15 does however see the control of apical wall growth as being the result of a “delicate balance between wall synthesis and wall lysis”, with some of the vesicles being transported to the apex containing lytic enzymes, to keep the wall at the apex in a plastic and extensible condition. Certainly in every case investigated chitinase activities can be detected in actively growing chitinous fungi. Examples include Mucor and Phycomyces species16–20,Neurospora crassa
21,22Aspergillus nidulans
9,23, Saccharomyces cerevisiae
24 and Candida albicans
25.
