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Chitin synthases are very important enzymes for chitin synthesis in various species, which makes them a specific target of insecticides. In the present study, the function of the chitin synthase A (CHSA) gene isolated from Mythimna separate is investigated. The majority of dsMysCHSA treated larvae (89.50%) exhibit lethal phenotypes, including three phenotypes with severe cuticle deformations. The dsMysCHSA treatment in adult females affects oogenesis, and significantly reduce the ovary size and the oviposition number compared with controls. To determine how MysCHSA affects female fecundity, combined analyses of RNA-sequencing (RNA-Seq) transcriptome and TMT proteome (tandem mass tags) data in M. separata after treatment with MysCHSA-RNAi is performed. The differentially expressed proteins and genes affect fecundity-related proteins, energy metabolism, fatty acid metabolism, amino sugars, and nucleotide sugar metabolism pathways. Taken together, these results suggest that MysCHSA acts on M. separata ecdysis and fecundity, and has the potential as a target gene for pest control., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chs5p is a component of the exomer, a coat complex required to transport the chitin synthase Chs3p from the trans-Golgi network to the plasma membrane. The Chs5p N-terminal region exhibits fibronectin type III (FN3) and BRCT domains. FN3 domains are present in proteins that mediate adhesion processes, whereas BRCT domains are involved in DNA repair. Several fungi--including Schizosaccharomyces pombe, which has no detectable amounts of chitin--have proteins similar to Chs5p. Here we show that the FN3 and BRCT motifs in Chs5p behave as a module that is necessary and sufficient for Chs5p localization and for cargo delivery. The N-terminal regions of S. cerevisiae Chs5p and S. pombe Cfr1p are interchangeable in terms of Golgi localization, but not in terms of exomer assembly, showing that the conserved function of this module is protein retention in this organelle and that the interaction between the exomer components is organism-specific.

A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin biosynthesis) has been established. Immuno-electronmicroscopical studies of purified chitosomes, gained from a yeast strain carrying a chitin-synthase gene fused to that for GFP (green-fluorescence protein), has led to the in situ localization of chitin-synthase-GFP molecules within chitosomes.

The molecular underpinnings of the oocyte-to-embryo transition are poorly understood. Here we show that two protein tyrosine phosphatase-like (PTPL) family proteins, EGG-4 and EGG-5, are required for key events of the oocyte-to-embryo transition in Caenorhabditis elegans. The predicted EGG-4 and EGG-5 amino acid sequences are 99% identical and their functions are redundant. In embryos lacking EGG-4 and EGG-5, we observe defects in meiosis, polar body formation, the block to polyspermy, F-actin dynamics, and eggshell deposition. During oogenesis, EGG-4 and EGG-5 assemble at the oocyte cortex with the previously identified regulators or effectors of the oocyte-to-embryo transition EGG-3, CHS-1, and MBK-2 [1, 2]. All of these molecules share a complex interdependence with regards to their dynamics and subcellular localization. Shortly after fertilization, EGG-4 and EGG-5 are required to properly coordinate a redistribution of CHS-1 and EGG-3 away from the cortex during meiotic anaphase I. Therefore, EGG-4 and EGG-5 are not only required for critical events of the oocyte-to-embryo transition but also link the dynamics of the regulatory machinery with the advancing cell cycle.

The pathways responsible for cell wall polysaccharide biosynthesis are vital in eukaryotic microorganisms. The corresponding synthases are potential targets of inhibitors such as fungicides. Despite their fundamental and economical importance, most polysaccharide synthases are not well characterized, and their molecular mechanisms are poorly understood. With the example of Saprolegnia monoica as a model organism, we show that chitin and (1-->3)-beta-d-glucan synthases are located in detergent-resistant membrane microdomains (DRMs) in oomycetes, a phylum that comprises some of the most devastating microorganisms in the agriculture and aquaculture industries. Interestingly, no cellulose synthase activity was detected in the DRMs. The purified DRMs exhibited similar biochemical features as lipid rafts from animal, plant, and yeast cells, although they contained some species-specific lipids. This report sheds light on the lipid environment of the (1-->3)-beta-d-glucan and chitin synthases, as well as on the sterol biosynthetic pathways in oomycetes. The results presented here are consistent with a function of lipid rafts in cell polarization and as platforms for sorting specific sets of proteins targeted to the plasma membrane, such as carbohydrate synthases. The involvement of DRMs in the biosynthesis of major cell wall polysaccharides in eukaryotic microorganisms suggests a function of lipid rafts in hyphal morphogenesis and tip growth.

Little precise information is available on the systematics, genetics, ecology and epidemiology of yeasts of the genus Malassezia from different animal species. In the present study, one hundred and four isolates of Malassezia (lipid dependent or non-lipid dependent) from dogs were characterized by their chitin synthase 2 gene (CHS2), and the large subunit (LSU) and the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA sequences, and compared genetically with well-defined reference strains of Malassezia pachydermatis and heterologous species, including Malassezia furfur and Candida albicans. For each locus examined, three main sequence types (i.e. A, B and C) represented all of the 104 isolates, which were designated as genotypes A, B and C, respectively. A fourth, minor sequence type was also defined for the ITS-1. The nucleotide differences among genotypes was consistent with the magnitudes of intraspecific variability reported in previous studies. The genetic analysis of the sequence data sets (for individual loci) showed that all Malassezia genotypes clustered (with moderate to strong support) with the reference sequences of M. pachydermatis to the exclusion of the outgroups M. furfur and C. albicans. The present study reveals that multiple genetic variants of M. pachydermatis occur on dogs. The multilocus approach employed herein provides a foundation for future investigations of M. pachydermatis from other animals and humans, and their ecology and epidemiology.

Labeled UDP-GlcNAc and chitooligosaccharides should be powerful tools for studies of N-acetylglucosaminyltransferase such as chitin synthases. We describe here a rapid, inexpensive and a common strategie for the chemoenzymatic synthesis of uridine 5'-diphospho-N-[(2)H]-acetyl-glucosamine and the chemical preparation of N-[(2)H]-acetyl chitooligosaccharides (from 2 to 5 mers). Deuterated UDP-GlcNAc analogue was tested as chitin synthase substrate and it exhibited an incorporation level in chitin as the natural substrate. Deuterium labeling of carbohydrates present different advantages: it is a stable isotope and allows glycosyltransferase mechanism studies by a mass spectrometry approach.

Conserved polypeptides of the chitin synthase genes UmCHS3 and UmCHS6 from the phytopathogenic fungus Ustilago maydis were utilized as immunogens to obtain polyclonal antibodies that were purified by affinity procedures. Because of their similarities at the regions encoded by either polypeptide, it was concluded that anti-Chs3 antibodies recognized both Chs3 and Chs4 chitin synthases, whereas anti-Chs6 antibodies recognized Chs6 and Chs8 polypeptides. These antibodies were used to analyze the localization of the corresponding chitin synthases in U. maydis cells, using both indirect immunofluorescence microscopy and immunoelectron microscopy with colloidal-gold-labeled secondary antibodies. It was observed that chitin synthase proteins were accumulated both in the surface and in the cytoplasm of the fungal cells. Electron microscopy images revealed the accumulation of clusters of gold particles in vesicles, providing evidence for the possible origin and destination of chitin synthases in the fungal cells.

A single chitin synthase gene is responsible for chitin production in the eggshells of the plant parasitic nematode Meloidogyne artiellia. In this paper we describe a functional analysis of this gene using RNAi as well as further analysis of two similar genes from the free-living nematode Caenorhabditis elegans. The parasitic life-style of M. artiellia required the development of a novel method for delivery of dsRNA to nematode eggs that may be of utility in other experimental systems. C. elegans chitin synthase genes were silenced by feeding nematodes bacteria expressing appropriate chitin synthase dsRNA from a plasmid vector, while M. artiellia egg masses were soaked in dsRNA solution. The results obtained demonstrated that the synthesis of chitin continues to take place in nematode eggs within the egg sac in the parasitic nematode, and that the removal of this activity affects egg development in both C. elegans and M. artiellia. The method described here provides a new way of investigating gene function in plant parasitic nematodes allowing the validity of potential target genes for novel control methods to be assessed. Furthermore, since intact egg cells within the gelatinous matrix of M. artiellia are permeable to dsRNA, eggs of other nematodes may also be similarly permeable to dsRNA and therefore amenable to use with dsRNAi.

Chitin synthase III is essential for the increase in chitin level and for cell integrity in cells lacking Gas1p, a beta(1,3)-glucanosyltransferase. In order to discover whether the upregulation of chitin synthesis proceeds through the canonical transport and activation pathway of Chs3p or through an alternative one, here we studied the effects of the inactivation of the GAS1 and CHS4-5-6-7 genes. All the double-null mutants showed a temperature-sensitive cell lysis phenotype that could be suppressed by the presence of an osmotic stabilizer. In liquid YEPD at 30 degrees C, chs4 delta gas1 delta, chs5 delta gas1 delta, chs6 deltagas1 delta and chs7 delta gas1 delta mutants were unable to grow, whereas they grew very slowly in minimal medium and showed low viability. High osmolarity suppressed the defective phenotype and restored growth. In chs4 gas1, chs5 gas1 and chs7 gas1, chitin levels did not increase and were reduced to only 10%, while in chs6 gas1 the value of gas1 was reduced to 20-40%. To investigate at which level the upregulation of chitin synthesis could occur, mRNA levels were monitored. The expression of CHS4-5-6-7 did not change significantly in gas1 delta. In strains expressing HA-tagged forms, the localization of Chs3p and Chs5p was examined. In the gas1 mutant the fluorescence pattern was affected and the proteins appeared abnormally present in the bud. The results indicate that: (a) the function of the CHS4-7 genes is required for chitin hyperaccumulation in gas1 mutant and for cell integrity; (b) homologous genes do not replace their function; (c) the regulation of CHS4-7 genes does not occur at transcriptional level. Control of the position of chitin synthesis could be important in protecting the bud from lysis., (Copyright 2002 John Wiley & Sons, Ltd.)

Yeast cells were permeabilized by incubation in 0.8 M sorbitol followed by suspension in dilute buffer. A preincubation with 2-mercaptoethanol was also included for optimal permeabilization. More than 90% of the treated cells were stainable with methylene blue. Determinations of cell wall-synthesizing enzymes (beta(1 --> 3)glucan and chitin synthases) and cytosolic enzymes in permeabilized cells yielded similar or higher activities than those in cell extracts. With chitin synthase III, the activity obtained with cells was 4- to 6-fold higher than in membrane preparations. Little protein leaks from the cells during permeabilization; yet the cells appear to be readily permeable to substrates and even proteins. Thus, these preparations may be of wide use for the study of enzymes and of biological processes in situ.

Permeabilization of yeast and other fungal cells by osmotic shock enabled the in situ assays of intracellular plasma membrane-bound enzymes, such as beta-1,3-glucan synthase, chitin synthase, and Na(+)/K(+) ATPase as well as the soluble, cytoplasmic enzymes, such as lactate dehydrogenase and alpha-glucosidase. The permeabilization was accomplished by rapid changes in osmolarity of the washing buffer at 0 degrees C whereby 0.5-3.5 M glycerol, sorbitol, and/or mannitol and/or 1 M KCl could be used as the osmolytes. No appreciable leakage of intracellular proteins occurred during the permeabilization procedure. The described procedure caused practically complete cell permeabilization while avoiding treatments with organic solvents, detergents, and other xenobiotics currently used for the permeabilization of microbial cells.

The cell wall of fungal cells is important for cell integrity and cell morphogenesis and protects against harmful environmental conditions. The yeast cell wall is a complex structure consisting mainly of mannoproteins, glucan, and chitin. The molecular mechanisms by which the cell wall components are synthesized and transported to the cell surface are poorly understood. We have identified and characterized two homologous yeast proteins, Sbe2p and Sbe22p, through their suppression of a chs5 spa2 mutant strain defective in chitin synthesis and cell morphogenesis. Although sbe2 and sbe22 null mutants are viable, sbe2 sbe22 cells display several phenotypes indicative of defects in cell integrity and cell wall structure. First, sbe2 sbe22 cells display a sorbitol-remediable lysis defect at 37 degrees C and are hypersensitive to SDS and calcofluor. Second, electron microscopic analysis reveals that sbe2 sbe22 cells have an aberrant cell wall structure with a reduced mannoprotein layer. Finally, immunofluorescence experiments reveal that in small-budded cells, sbe2 sbe22 mutants mislocalize Chs3p, a protein involved in chitin synthesis. In addition, sbe2 sbe22 diploids have a bud-site selection defect, displaying a random budding pattern. A Sbe2p-GFP fusion protein localizes to cytoplasmic patches, and Sbe2p cofractionates with Golgi proteins. Deletion of CHS5, which encodes a Golgi protein involved in the transport of Chs3p to the cell periphery, is lethal in combination with disruption of SBE2 and SBE22. Thus, we suggest a model in which Sbe2p and Sbe22p are involved in the transport of cell wall components from the Golgi apparatus to the cell surface periphery in a pathway independent of Chs5p.

In the present study, to confirm Microsporum canis infection rapidly, we detected the chitin synthase gene 1 (CHS1) gene of M. canis in the hair and skin samples of four dogs with dermatophytosis. Amplification of the DNAs in the four samples with CHS1 primers yielded fragments of about 620-bp. Nucleotide sequence analysis of the CHS1 gene fragments from samples and a reference strain of M. canis gene showed more than 99% similarity. The method presented in this study can rapidly detect the DNA of M. canis in skin scrapings, and we anticipate that it will be a useful microbiological tool for the diagnosis of M. canis infections in animals and humans.

The presence of non-fibrillar alpha-chitin in cellulosic fungi (class Oomycetes) poses intriguing questions as to its role, subcellular localization and evolutionary significance. Previous studies reported on the similarity of chitin synthase from Saprolegnia monoica with that of other fungi. The present work describes important dissimilarities. There was no evidence that the chitin synthase of S. monoica was present in small low-density vesicles (chitosomes). Chitin synthase sedimented with membranous components of high specific gravity (sp. gr. 1.177) that could be partially but distinctly separated from membranes harbouring most of the 1,3-beta-glucan synthase in the cell (sp. gr. 1.158). In contrast to other fungi, the chitin synthase from S. monoica was greatly stimulated by digitonin: both membrane-bound and dissociated chitin synthase showed little activity in the absence of digitonin. As in other fungi, the chitin synthase from S. monoica was solubilized by digitonin and remained zymogenic after dissociation. However, unlike the enzyme from other fungi, the solubilized chitin synthase of S. monoica had a lower sedimentation coefficient, was not stimulated by phospholipids and was not inhibited by high concentrations of digitonin. Unlike the enzyme from Mucor rouxii, the solubilized chitin synthase from S. monoica did not bind to a cation exchanger. The enzyme was partially purified by four-step scheme that included sucrose density-gradient centrifugation, a single passage through a strong anion exchanger and two consecutive passages through a weak anion exchanger. The final preparation contained five to seven polypeptide bands that cochromatographed with the chitin synthase activity, some of which may be part of a presumed chitin synthase macromolecular complex.

PCR was used to amplify fragments corresponding to the chitin synthase (CHS) genes from the Oomycetes Saprolegnia monoica, Phytophthora capsici and Achlya ambisexualis, utilizing as primers, oligonucleotides designed from the conserved region of CHS genes of chitinous fungi. Chitin synthase homologues were found in the three cellulosic fungi. The chitin synthase 2 gene (CHS2) from S. monoica was cloned, sequenced and characterized. The amino acid sequence deduced from the CHS2 genomic DNA revealed several domains, corresponding to the catalytic domains and polypeptide signatures, of high identity with CHS genes from chitinous fungi. Existence of a CHS gene family in S. monoica was supported by the identification of two CHS sequences among the PCR products, the localization of CHS homologues on two chromosomes, and the detection of two transcripts in mycelia and protoplasts. Polyclonal anti-chitin synthase antibodies raised against the N-terminal and the neutral fragments of the CHS2 products revealed, respectively, two and four proteins in membrane fractions and a truncated active form in entrapped product. The overall comparison of the structure and organization of CHS genes indicates that in spite of their divergent evolution, Oomycetes and chitinous fungi have evolved with conserved chitin synthase systems.

Chitin is an essential structural component of the yeast cell wall whose deposition is regulated throughout the yeast life cycle. The temporal and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomyces cerevisiae by localization of the putative catalytic subunit of chitin synthase III, Chs3p, and its regulator, Chs5p. Immunolocalization of epitope-tagged Chs3p revealed a novel localization pattern that is cell cycle-dependent. Chs3p is polarized as a diffuse ring at the incipient bud site and at the neck between the mother and bud in small-budded cells; it is not found at the neck in large-budded cells containing a single nucleus. In large-budded cells undergoing cytokinesis, it reappears as a ring at the neck. In cells responding to mating pheromone, Chs3p is found throughout the projection. The appearance of Chs3p at cortical sites correlates with times that chitin synthesis is expected to occur. In addition to its localization at the incipient bud site and neck, Chs3p is also found in cytoplasmic patches in cells at different stages of the cell cycle. Epitope-tagged Chs5p also localizes to cytoplasmic patches; these patches contain Kex2p, a late Golgi-associated enzyme. Unlike Chs3p, Chs5p does not accumulate at the incipient bud site or neck. Nearly all Chs3p patches contain Chs5p, whereas some Chs5p patches lack detectable Chs3p. In the absence of Chs5p, Chs3p localizes in cytoplasmic patches, but it is no longer found at the neck or the incipient bud site, indicating that Chs5p is required for the polarization of Chs3p. Furthermore, Chs5p localization is not affected either by temperature shift or by the myo2-66 mutation, however, Chs3p polarization is affected by temperature shift and myo2-66. We suggest a model in which Chs3p polarization to cortical sites in yeast is dependent on both Chs5p and the actin cytoskeleton/Myo2p.

This study investigated the effect of the chitin synthetase inhibitors, the nikkomycins (NZ and NZ+NX), on Candida albicans adhesion to buccal epithelial cells (BECs) in vitro. The effect was expressed in reduced chitin synthetase activity and chitin content of fungal cells. In vitro adhesion assays to BECs of Candida exposed to NZ and NZ+NX revealed reduced adhesion values. Light, scanning and transmission electron microscopy (SEM, TEM) of NZ-treated and untreated micro-organisms showed changed fungal morphology and reduced adherence of the treated yeasts. Scanning electron microscopy of NZ-treated C. albicans labelled with gold-conjugated wheatgerm agglutinin (WGA) revealed less labelling than in the untreated organisms. A close contact between the fungus and the epithelial cell at a site with intense WGA-gold labelling was noted in TEM experiments. The data point to the involvement of chitin in the adhesion of C. albicans to epithelial cells.

The deposition of the polysaccharide chitin in the Saccharomyces cerevisiae cell wall is temporally and spatially regulated. Chitin synthase III (Chs3p) synthesizes a ring of chitin at the onset of bud emergence, marking the base of the incipient bud. At the end of mitosis, chitin synthase II (Chs2p) deposits a disk of chitin in the mother-bud neck, forming the primary division septum. Using indirect immunofluorescence microscopy, we have found that these two integral membrane proteins localize to the mother-bud neck at distinct times during the cell cycle. Chs2p is found at the neck at the end of mitosis, whereas Chs3p localizes to a ring on the surface of cells about to undergo bud emergence and in the mother-bud neck of small-budded cells. Cell synchronization and pulse-chase experiments suggest that the timing of Chs2p localization results from cell cycle-specific synthesis coupled to rapid degradation. Chs2p degradation depends on the vacuolar protease encoded by PEP4, indicating that Chs2p is destroyed in the vacuole. Temperature-sensitive mutations that block either the late secretory pathway (sec1-1) or the internalization step of endocytosis (end4-1) also prevent Chs2p degradation. In contrast, Chs3p is synthesized constitutively and is metabolically stable, indicating that Chs2p and Chs3p are subject to different modes of regulation. Differential centrifugation experiments show that a significant proportion of Chs3p resides in an internal compartment that may correspond to a vesicular species called the chitosome (Leal-Morales, C.A., C.E. Bracker, and S. Bartnicki-Garcia. 1988, Proc. Natl. Acad. Sci. USA. 85:8516-8520; Flores Martinez, A., and J. Schwencke. 1988. Biochim. Biophys. Acta. 946:328-336). Fractionation of membranes prepared from mutants defective in internalization (end3-1 and end4-1) indicate that the Chs3p-containing vesicles are endocytically derived. Collectively, these data suggest that the trafficking of Chs2p and Chs3p diverges after endocytosis; Chs3p is not delivered to the vacuole, but instead may be recycled.

In the light of recent genetic advances, some results regarding chitin biosynthetic activities are reviewed in this paper. Genes coding for distinct enzymes displaying chitin synthetase activities have been characterized in Saccharomyces cerevisiae as well as in other fungal species including Candida albicans. Several activities seem to exist in the cells: (i) one zymogenic, located in cytoplasmic vesicles called chitosomes, although the presence of other types of vesicles with zymogenic activity cannot be completely discarded, and (ii) plasma membrane associated activities (the active enzyme and probably two distinct pools of zymogenic activity). Possible relationships between these activities, if any, remain to be determined. These multiplicity of enzymes is not surprising taking into account that chitin biosynthesis is required during very well defined temporal and spatial events of the cell cycle. A general repair function for one of the chitin biosynthetic activities is proposed as a possible salvage mechanism to warrant cell survival after wall damage has been caused, since chitin appears to be the most suitable polymer to carry out this function due to its particular physico-chemical properties.

A novel, potent, semisynthetic pneumocandin, L-733,560, was used to isolate a resistant mutant in Saccharomyces cerevisiae. This compound, like other pneumocandins and echinocandins, inhibits 1,3-beta-D-glucan synthase from Candida albicans (F.A. Bouffard, R.A. Zambias, J. F. Dropinski, J.M. Balkovec, M.L. Hammond, G.K. Abruzzo, K.F. Bartizal, J.A. Marrinan, M. B. Kurtz, D.C. McFadden, K.H. Nollstadt, M.A. Powles, and D.M. Schmatz, J. Med. Chem. 37:222-225, 1994). Glucan synthesis catalyzed by a crude membrane fraction prepared from the S. cerevisiae mutant R560-1C was resistant to inhibition by L-733,560. The nearly 50-fold increase in the 50% inhibitory concentration against glucan synthase was commensurate with the increase in whole-cell resistance. R560-1C was cross-resistant to other inhibitors of C. albicans 1,3-beta-D-glucan synthase (aculeacin A, dihydropapulacandin, and others) but not to compounds with different modes of action. Genetic analysis revealed that enzyme and whole-cell pneumocandin resistance was due to a single mutant gene, designated etg1-1 (echinocandin target gene 1), which was semidominant in heterozygous diploids. The etg1-1 mutation did not confer enhanced ability to metabolize L-733,560 and had no effect on the membrane-bound enzymes chitin synthase I and squalene synthase. Alkali-soluble beta-glucan synthesized by crude microsomes from R560-1C was indistinguishable from the wild-type product. 1,3-beta-D-Glucan synthase activity from R560-1C was fractionated with NaCl and Tergitol NP-40; reconstitution with fractions from wild-type membranes revealed that drug resistance is associated with the insoluble membrane fraction. We propose that the etg1-1 mutant gene encodes a subunit of the 1,3-beta-D-glucan synthase complex.

Saccharomyces cerevisiae contains three chitin synthetases, Chs1, Chs2, and Chs3, performing different physiological functions but catalyzing the same reaction. It has been found that Ni2+ is a powerful inhibitor of Chs1 and Chs2 activity but has very little effect on the activity of Chs3, especially in the presence of Co2+. These results, together with the previous knowledge that Co2+ stimulates Chs2 and Chs3 but inhibits Chs1 and that the three synthetases differ in their pH optimum, have enabled us to formulate conditions for the specific determination of each synthetase in the presence of the others.

The effect of a lipase activity (EC 3.1.1.3) on the chitin synthetase from Candida albicans has been studied, both on the active and the trypsin activated enzyme. Removal of fatty acids from acylglycerides by lipase has an inhibitory effect on the activity as well as on the 'in vitro' activation process by trypsin in the membrane-bound enzyme and in the chitosomes. This would indicate that an adequate lipid environment is required for both the activation process and proper function of the synthetase activity.

In this paper we studied the transformation of a trophozoite into a cyst in Entamoeba, using E. invadens as a model. We had the following objectives: a) identification of precyst-specific proteins (P), by a monoclonal antibody against E. invadens and heterologous polyclonal antibodies against cellular (165) fractions of Mucar rouxii, which are chitin synthetase activity rich; and b) in vivo determination of the time required for the expression of activity of the chitin synthetase during encystment. We found P markers which are not found in either trophozoites nor cysts. Monoclonal F507 antibody recognized a 33 kDa protein in P and the polyclonal anti-16S antibodies reacted with a 90 kDa protein to P. Even though the 33 and 90 kDa proteins have a different molecular weight from the chitin synthetase described in fungi (57 kDa and 65 kDa) and yeasts (63 kDa), we conclude that these proteins are specific to P and that the 90 kDa one shares epitopes with chitosomal fractions of M. rouxii. Also, the mayor accumulation of alkali-resistant material, sensitive to chitinase, occurred during the formation of P, between 40 and 50 hours post incubation, during encystment. One may conclude that chitin polymers are synthesized during the P phase.

Investigations were carried out into the properties and localization of chitin synthase (EC 2.4.1.16) in connection with the formation of Ascaris suum egg-shells. The chitin synthase of eggs exhibited properties very similar to those found in other chitin-synthesizing organisms. It was stimulated by the presence of ATP, GlcNAc and Mg2+ with maximal activity at pH 7.0. In the phase of active chitin synthesis, during formation of the primary shell layers, chitin synthase was bound to the structures of the eggs. On completion of chitin synthesis in the eggs, the amount of the enzyme increased in the soluble fraction. Two forms of chitin synthase have been found in the eggs, namely an inactive (zymogenic) and an active form. Transformation of these two enzyme forms represents a key mechanism in the regulation of chitin synthesis in A. suum eggs.

Pretreatment of yeast protoplasts with concanavalin A, according to the method used by G. A. Scarborough for Neurospora (J. Biol. Chem. 250, 1106-1111, 1975), reinforced the plasma membranes, and helped to maintain their integrity during subsequent lysis of the protoplasts. After purification by centrifuging on a Renografin density gradient, practically intact membranes were obtained. Previous labeling of the protoplasts with 125I or with [3H]concanavalin A resulted in recovery of the radioactivity in the membrane fraction. The bulk of the chitin synthetase (chitin synthase; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase; EC 2.4.1.16) recovered in the gradient was also found In this fraction; in the zymogen form. About 20% of the activity sedimented in a plasma-membrane-free fraction at lower density. Glutaraldehyde inactivated chitin synthetase when it was added to a lysate, but not when applied to intact protoplasts. It is concluded that chitin synthetase is so oriented in the membrane that it is only accessible from the inside of the cell. These results confirm our previous hypothesis that the chitin synthetase zymogen is associated with the plasma membrane, a basic assumption for the explanation of localized activation of the enzyme and initiation of septum formation.

The gamma particles of Blastocladiella emersonii are 0.5-micron (diameter), electron dense, membrane-enclosed organelles in the cytoplasm of zoospores that have been reported (E.C. Cantino and G.L. Mills, in P. Lemke, ed., Viruses and Plasmids in Fungi, 1979, and R.B. Myers and E.C. Cantino, in A. Wolsky (ed.), Monographs in Developmental Biology, 1974) to store the enzyme chitin synthetase. These particles were isolated from zoospores, and the two major proteins were purified for an analysis of their composition and function. The lower-molecular-weight protein (apparent molecular weight, 41,000) was insoluble in aqueous buffers, had an unusual, very basic amino acid composition, and comprised the characteristic electron-dense inclusions seen in micrographs of sections of fixed and stained gamma particles. After dispersal of the gamma particle membranes with detergent, the higher-molecular-weight protein (apparent molecular weight, 43,000) and a third minor protein (apparent molecular weight, 45,000) sedimented through sucrose cushions with the 41 kilodalton inclusion body protein but were dissociated from it by sonication in buffer containing 7 M urea. Together, the two major proteins represent 60 to 70% of the total protein in the gamma particle and 2.9% of the total protein in zoospores. Tests with specific antisera showed that the two major proteins were not antigenically related, a result consistent with the differences in amino acid composition. When zoospore lysates were centrifuged in sucrose density gradients, the major gamma particle proteins and chitin synthetase activity migrated to regions of different density. Proteins from sporulating thalli and germinating zoospores were separated by gel electrophoresis, and the two major gamma particle proteins were detected by reaction with specific antisera after electrophoretic transfer to nitrocellulose filters. Neither protein could be found in growth phase cells; the appearance and disappearances of both proteins were correlated with the formation of the gamma particles during sporulation and their decay during zoospore germination. The results indicate that gamma particles do not store chitin synthetase in the proteinaceous inclusion, but an alternative function has not yet been identified.

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.

We describe an improved method for fractionating cell-free extracts of Saccharomyces cerevisiae to separate its membranous components by a combination of isopycnic and velocity sedimentations. These procedures were used to examine the subcellular distribution of chitin synthetase (chitin-UDP acetylglucosaminyltransferase; EC 2.4.1.16) in homogenates from exponentially growing walled cells of a wild-type strain of yeast. Chitin synthetase (Chs1) activity was mainly found in two distinct vesicle populations of nearly equal abundance but with markedly different buoyant densities and particle diameters. One population contained 45-65% of the total chitin synthetase and was identified as chitosomes because of microvesicular size (median diameter = 61 nm) and characteristic low buoyant density (1.15 g/ml); it also lacked 1,3-beta-glucan synthetase activity. The second population (35-55%) was identified as plasma membrane because of its high buoyant density (1.22 g/ml), large vesicle size (median diameter = 252 nm), and presence of vanadate-sensitive ATPase. This fraction cosedimented with the main peak of 1,3-beta-glucan synthetase. A third, minor population of chitin synthetase particles was also detected. Essentially all of the chitin synthetase in the two vesicle populations was zymogenic; therefore, we regard these vesicles as precursors of the final active form of chitin synthetase whose location in the cell has yet to be unequivocally determined.

Cell homogenates obtained from partially regenerated Saccharomyces cerevisiae protoplasts were fractionated by a procedure using a combination of continuous and discontinuous sucrose gradients, under experimental conditions that minimize possible artifacts due to centrifugation and resuspension. At least five different membranous organelle fractions (plasma membrane, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum-like structures and small-sized particulated structures) were isolated. Subcellular fractions were characterized by assaying established marker enzymes. Radioactive labelled [(U-3H]uracil) ribosomes were also used as a further characterization criterion of the rough endoplasmic reticulum. Comparative SDS-polyacrylamide gel electrophoresis of the protein constituents of the isolated membrane-bound organelles suggest that the polypeptide pattern could also be used as an additional marker for some of these structures. Finally, subcellular distribution of chitin synthase was determined using this fractionation procedure, and two partially zymogenic enzyme pools (one inside the cell associated to particles which sediments at high speed, and the second one associated to the plasma membrane) were found.