Chitosomes: past, present and future

José Ruiz-Herrera's discovery that chitin microfibrils could be made by a fungal extract paved the way for elucidating the intracellular location of chitin synthetase. In collaboration with Charles Bracker, chitosomes were identified as the major reservoir of chitin synthetase in fungi. Unique in size, buoyant density, and membrane thickness, chitosomes were found in a wide range of fungi. Their reversible dissociation into 16S subunits is another unique property of chitosomes. These 16S subunits are the smallest molecular entities known to retain chitin synthetase activity. Further dissociation leads to complete loss of activity. From studies with secretory mutants, yeast researchers concluded that chitosomes were components of the endocytosis pathway. However, key structural and enzymatic characteristics argue in favor of the chitosome being poised for exocytotic delivery rather than endocytotic recycling. The chitosome represents the main vehicle for delivering chitin synthetase to the cell surface. An immediate challenge is to elucidate chitosome ontogeny and the role of proteins encoded by the reported chitin synthetase genes in the structure or function of chitosomes. The ultimate challenge would be to understand how the chitosome integrates with the cell surface to construct the organized microfibrillar skeleton of the fungal cell wall.     

Sedimentation properties of chitosomes fromMucor rouxii

Summary This study was undertaken to assess the distribution and localization of chitin synthetase in a fungal cell and to evaluate the sedimentation behavior of chitosomes (microvesicular containers of chitin synthetase). Chitosomes were isolated from cell-free extracts of yeast cells ofMucor rouxii by rate-zonal and isopycnic sedimentation in sucrose density gradients. Because of their small size and low density, chitosomes were effectively separated from other subcellular particles. Rate-zonal sedimentation was a suitable final step for isolating chitosomes as long as ribosomes had been eliminated by enzymic digestion. By isopycnic centrifugation, chitosomes could be separated directly from a crude cell-free extract; they cosedimented with a sharp symmetrical peak of chitin synthetase at a buoyant density of d=1.14–1.15g/cm³; the only significant contaminants were particles of fatty acid synthetase complex. From such sedimentations, we estimated that 80–85% of the chitin synthetase activity in the cell-free extract was associated with chitosomes; the rest was found in two smaller peaks sedimenting at d=1.19–1.20 and d=1.21–1.22 (5–10%), and in the cell wall fraction (5–10%). By consecutive rate-zonal and isopycnic sedimentations, chitosome preparations with relatively few contaminating particles were obtained. Potassium/sodium phosphate buffer (pH 6.5)+MgCl₂ was the most effective isolation medium for chitosomes. Other buffers such as TRIS-MES+MgCl₂ led to massive aggregation of chitosomes and a change in sedimentation properties. This tendency of chitosomes to aggregate could explain why most of the chitin synthetase activity of a fungus is sometimes found associated with other subcellular structures,e.g., plasma membrane.     

Chitosomes from the wall-less “slime” mutant of Neurospora crassa

Cell-free extracts from the wall-less slime mutant of Neurospora crassa and the mycelium of wild type exhibit similar chitin synthetase properties in specific activity, zymogenicity and a preferential intracellular localization of chitosomes. The yield of chitosomal chitin synthetase from sline cells was essentially the same irrespective of cell breakage procedure (osmotic lysis or ballistic disruption) —an indication that chitosomes are not fragments of larger membranes produced by harsh (ballistic) disruption procedures. The plasma membrane fraction, isolated from slime cells treated with concanavalin A, contained only a minute portion of the total chitin synthetase of the fungus. Most of the activity was in the cytoplasmic fraction; isopycnic sedimentation of this fraction on a sucrose gradient yielded a sharp band of chitosomes with a buoyant density=1.125 g/ cm³. Approximately 76% of the total chitin synthetase activity of the slime mutant was recovered in the chitosome band. Because of their low density, chitosomes could be cleanly separated from the rest of the membranous organelles of the fungus. Apparently, the lack of a cell wall in the slime mutant is not due to the absence of either chitosomes or zymogenic chitin synthetase.Abbreviations Con A concanavalin A - d buoyant density in g/cm³ - GlcNAc N-acetyl-D-glucosamine - MES 2-[N-morpholino]ethanesulfonic acid - UDP-GlcNAc uridine diphosphate N-acetyl-D-glucosamine     

Chitin synthetase activity is bound to chitosomes and to the plasma membrane in protoplasts of Saccharomyces cerevisiae

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacuolar proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40-110 nm; buoyant density (d) = 1.146 g/cm3). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm3) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with 3H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100-250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.     

Protein composition of purified chitosomes ofMucor rouxii

Different centrifugation conditions were compared for purification of chitosomes (microvesicles containing chitin synthetase). Isopycnic centrifugation of crude chitosome samples from yeast cells ofMucor rouxii on sucrose density gradients, in a vertical rotor, yielded chitosomes of higher purity than before. About 90–96% of the chitin synthetase in purified chitosomes was zymogenic. We estimated that the chitosome population of the yeast form ofM. rouxii comprises a miniscule portion (0.17%) of the total cell protein. The polypeptide composition of purified chitosomes was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Prominent polypeptide bands were found at 16, 18, 28, 30, 32, 44, 47, and 55 kDa. The 55-kDa polypeptide was most conspicuous. There were also minor bands at 25, 26, 42, 60, 67, and 80 kDa. Our findings show that highly purified populations of chitosomes consist of microvesicles with a characteristic size range, buoyant density, and a complex polypeptide composition.     

Chitin synthetase activity is bound to chitosomes anf to the plasma membrane in protoplasts of Saccharomyces cerevisiae

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacoular proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40–110 nm; bouyant density (d) = 1.146 g/cm³). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm³) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with ³H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100–250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.     

Chitosomes and chitin synthetase in the asexual life cycle of Mucor rouxii: spores, mycelium and yeast cells

To help understand the subcellular machinery responsible for cell wall formation in a fungus, we determined the abundance and subcellular distribution of chitin synthetase (chitin synthase, EC 2.4.1.16) and chitosomes in the asexual life cycle of Mucor rouxii. Cell-free extracts of ungerminated sporangiospores, hyphae/mycelium in exponential and stationary phase, and yeast cells were fractionated by isopycnic centrifugation in sucrose density gradients. The total amount of chitin synthetase per cell increased exponentially during aerobic germination of spores. In all developmental stages, the profile of chitin synthetase activity encompassed a broad range of sucrose density (d = 1.12-1.22) with two distinct zones: a low-density chitosome zone (d = approx. 1.12-1.16) and a high-density, mixed-membrane zone (d = approx. 1.16-1.22). Chitosomes were a major reservoir of chitin synthetase in all stages of the life cycle, including ungerminated spores. Two kinds of chitin synthetase profiles were recognized and correlated with the growth state. In nongrowing cells (ungerminated sporangiospores and stationary-phase mycelium), the profile was skewed toward lower densities with a sharp chitosome peak at d = 1.12-1.13. In actively growing cultures (aerobic mycelium or anaerobic yeast cells), the entire profile of chitin synthetase was displaced toward higher densities; the average buoyant density of chitosomes was higher (d = 1.14-1.16), and more chitin synthetase was associated with denser (d = 1.16-1.23) membrane fractions. In all life cycle stages, chitosomal chitin synthetase was almost completely zymogenic. In contrast to the enzyme from spores or from growing cells, samples of chitosomal chitin synthetase from stationary-phase mycelium were unstable and contained a high proportion of larger vesicles in addition to the typical microvesicles. The presence of chitosomes in ungerminated spores indicates that these cells are poised to begin synthesizing somatic (= vegetative) cell walls at the onset of germination. The increased buoyant density of chitosomes in actively growing cultures suggests that the composition of these microvesicles changes significantly as they mobilize chitin synthetase to the cell surface.     

Separation of chitosomal chitin synthetase from cell-free extracts ofNeurospora crassa “Slime” variant agglutinated with concanavalin A

Cell-free extracts of the wall-less slime variant ofNeurospora crassa were treated with concanavalin A (Con A); this treatment caused a massive agglutination of the particulate structures in the cell-free homogenate, although most (73%) of the chitin synthetase initially present in the cell-free extract remained in the supernatant obtained after sedimentation of the lectin-flocculated material. This chitin synthetase showed the sedimentation properties of chitosomes (unique microvesicular structures) and failed to bind [³H]Con A. A significant percentage (42%) of the chitin synthetase activity associated with the Con A-flocculated material probably corresponds to mechanically trapped chitosomes, whereas the rest of the activity in the Con A-agglutinated material might be a genuine part of the flocculated membranes and could represent a physiologically distinct form of the enzyme.     

Properties of chitin synthetase in isolated chitosomes from yeast cells of Mucor rouxii.

Chitin synthetase was isolated and purified 120-fold from the supernatant fraction (54,500 X g) of broken yeast cells of Mucor rouxii. The purified preparations consisted mainly of chitin synthetase particles (chitosomes) with an average size larger than 7 X 10(6) daltons (by gel filtration) and an average sedimentation coefficient of 105 S. The samples also contained other enzyme complexes (fatty acid synthetase, pyruvate dehydrogenase, and, depending on method, ribosomes). Nearly all of the chitosomal chitin synthetase occurred in a zymogenic form that required proteolytic activation. In most properties, the chitosomal enzyme was similar to crude enzyme (54,000 X g sediment): kinetics, activation by proteases, response to metals, stimulation by N-acetylglucosamine, and inhibition by polyoxin or UDP. One mamor difference was the much greater stability of the chitosomal chitin synthetase zymogen against spontaneous activation and destruction. Product (chitin microfibril) and enzyme (chitin synthetase) remained associated in a complex that was readily separated by centrifugation.     

Chitosomes lack concanavalin-A-binding sites

Whether intact or dissociated with digitonin, chitosomes isolated from the fungusMucor rouxii lack the ability to bind concanavalin A. The absence of external or internal concanavalin A-binding sites distinguishes the chitosome membrane no only from plasma membrane but also from membranes of other organelles (endoplasmic reticulum, mitochondrion, vacuole). This differential binding ability was used to partially separate chitosomal chitin synthetase from major membranes in a crude cell-free extract ofM. rouxii.     

Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells ofCandida albicans

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) fromC. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen,in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processesin vivo andin vitro or, alternatively, that both enzyme activities (activein vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml^–1 of digitonin and 64 µg ml^–1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 µg ml^–1 of protease, whereas activity strongly decreases in the presence of 64 µg ml^–1 of trypsin. Digitonin does not produce zymogen activationper se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.     

An efficient preparative isopycnic flotation method for the isolation of chitosomes

Summary Chitin synthetase, a key enzyme in fungal cell wall biosynthesis, is located in chitosomes (microvesicles). To produce large quantities of chitosomes for immunochemical and biochemical characterization, we developed a two-step purification procedure in which isopycnic sucrose density gradients were centrifuged at ultra-high gravitational forces (fixed-angle rotor at 361,000×g R_av). Chitosomes from yeast cells ofMucor rouxii were separated from the soluble proteins and from the larger membranes by isopycnic centrifugation of the cell-free extract. The resulting crude chitosome sample was adjusted to a higher sucrose concentration, and a sucrose gradient was layered over the sample. Upon recentrifugation, the chitosomes moved up into the gradient and equilibrated at their buoyant density (1.15–1.16). This accelerated flotation separated contaminating particles of higher buoyant density (larger vesicles, ribosomes, and other miniorganelles) and yielded a large population of microvesicles with a mean diameter of 48.9±13.8 nm. This preparation contained vesicles essentially free of other particulate contaminants; more than 99% of the vesicles were smaller than 100 nm. When required, an additional velocity centrifugation step was added to remove the larger vesicles from the chitosome samples. This streamlined method for chitosome isolation was much simpler and faster than earlier isolation procedures, gave a high yield of functional chitosomes, and made the large scale isolation of these organelles possible.     

The co-ordination of chitosan and chitin synthesis in Mucor rouxii

Chitin synthetase preparations from cell walls and chitosomes of the fungus Mucor rouxii were tested for their ability to synthesize chitosan when incubated with uridine diphosphate N-acetyl-D-glucosamine in the presence of chitin deacetylase. The most effective chitin synthetase preparation was one dissociated from cell walls with digitonin. The rate of chitosan synthesis by the wall-dissociated chitin synthetase was about three times that of an equivalent amount of cell walls. The chitosan-synthesizing ability of chitosomes was relatively low, but was more than tripled by treatment with digitonin. Presumably, digitonin improves chitosan yields of dissociating chitin synthetase. The dissociated enzyme would produce dispersed chitin chains that could be attacked by chitin deacetylase before they have time to crystallize into microfibrils. The regulation of chitin and chitosan syntheses in vivo may be determined by the organization of chitin synthetase molecules at the cell surface. Those molecules that remain organized as a complex, similar if not identical to that found in chitosomes, would produce mainly chitin. Chitosan would be preferentially produced by chitin synthetase molecules which are dispersed upon reaching the cell surface.     

Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway.

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.     

Stabilization of chitin synthetase and purification of chitosomes from several mycelial Mucorales

Stability of chitin synthetase in cell-free extracts from mycelial fungi was markedly improved by the presence of sucrose in the homogenization media. Breakage of mycelium in sucrose-containing buffer yielded enzyme preparations from which chitosomal chitin synthetase could be purified by a procedure involving ammonium sulfate precipitation, gel filtration and centrifugation in sucrose density gradients. Purified chitosomes catalyzed the synthesis of chitin microfibrils in vitro upon incubation with substrate and activators. Chitosomal chitin synthetase from the filamentous form of M. rouxii was similar to the enzyme from yeast cells, except for the poorer stability and diminished sensitivity to GlcNAc activation of the former.     

Purification and properties of the polymeric fatty acid synthetase from a filamentous fungus.

Fatty acid synthetase was purified from the filamentous fungus, Aspergillus fumigatus to a specific activity of 4000--5000 munits/mg protein. Its purity was established by its appearance in electron micrographs, on sodium dodecyl sulphate polyacrylamide gels and by analytical ultracentrifugation, and also by its behaviour upon sucrose gradient centrifugation. This enzyme comprises two large polypeptides with molecular weights of 190 000 and 186 000. Evidence from electron microscopy indicates that it consists of three equivalent loops of protein. It dissociates into different-sized circular subunits on ageing or upon dissolution in buffer of low ionic strength. Differences in properties between this fungal synthetase and that found in yeast have been noted and relate, for example, to inhibition by acetyl CoA and malonyl-CoA, cold-lability and pH optimum. The synthetase from A. fumigatus, purified by different procedures, consistently exists in two forms of similar specific activity, with sedimentation coefficients approx. 40 S and 60 S. Synthetase activity present in crude extracts has been identified as a very heavy component with sedimentation coefficient greater than 100 S.     

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.     

Activation of chitin synthetase in permeabilized cells of a Saccharomyces cerevisiae mutant lacking proteinase B.

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 procedures, we showed that increases in permeability and activation are distinct processes. Activation was inhibited by the protease inhibitors antipain and leupeptin, but by pepstatin or chymostatin. Metal chelators were also inhibitory, and their effect was reversed by the addition of Ca2+ but not by Mg2+. Antipain added together with Ca2+ after incubation of the cells in the presence of a chelating agent prevented reversal of inhibition, a result that was interpreted as indicating that antipain acts either on the same step affected by Ca2+ or on a subsequent step. Efforts to obtain activation in cell-free extracts were unsuccessful, but it was possible to extract the synthetase, once activated, by breaking permeabilized cells with glass beads. Treatment of the cell-free extracts with trypsin led not only to increased activity of chitin synthetase, but also to a change in the pH-activity curve and a diminished requirement by the enzyme for free N-acetylglucosamine. These observations suggest that the modification undergone by the synthetase during endogenous activation is different from that brought about by trypsin treatment.     

A nonradioactive,high throughput assay for chitin synthase activity

Wheat germ agglutinin (WGA) binds with high affinity and specificity to several sites on chitin polymers. Based on these properties we have modified and adapted a previously patented (U.S. patent 5,888,757) nonradioactive, high throughput screening assay for antimicrobial agents, making it suitable as a quantitative enzymatic assay for the activity of individual chitin synthase isozymes in yeast. The procedure involves binding of synthesized chitin to a WGA-coated surface followed by detection of the polymer with a horseradish peroxidase-WGA conjugate. Horseradish peroxidase activity is then determined as an increment in absorbance at 600 nm. Absorbance values are converted to amounts of chitin using acid-solubilized chitin as a standard. The high sensitivity (lower limit of detection about 50 ng chitin), low dispersion (lower than 10%), and high throughput (96-well microtiter plate format) make this assay an excellent substitute for the conventional radioactive chitin synthase assay in cell-free extracts. We have applied this method to the differential assay of chitin synthase activities (Chs1, Chs2, and Chs3) in cell-free extracts of Saccharomyces cerevisiae. Analysis of Chs3 activity in chitosomal and plasma membrane fractions revealed that Chs3 in the plasma membrane fraction is about sixfold more active than in the chitosome.     

Alterations in the vesicular pattern and wall growth ofPhycomyces induced by the calcium ionophore A 23187

Summary Hyphal elongation, chitin synthesis in vivo, and invertase secretion inPhycomyces blakesleeanus were all inhibited almost instantly by the addition of 5–10 M calcium ionophore A 23187. Protein biosynthesis was inhibited in these conditions by 30–50%. The ionophore did not affect cell respiration for at least 40 min. Effect on chitin biosynthesis was not due to alterations of the chitin synthetase levels or its activity; nor to impairement in GlcNAc metabolism. In drug-treated cells the number of apical vesicles was severely reduced even at very short periods of incubation, and these low numbers remained constant for at least 60 min of incubation with the ionophore. We suggest that the ionophore collapses the cellular calcium gradient and/or interferes with the normal electrical transhyphal current. As a consequence, formation and migration of apical vesicles are inhibited. These results are further evidence of the role of vesicles in fungal tip growth and exhibit the fact that active chitin synthetase is short-lived in vivo demanding its continuous supply by chitosomes to the cell surface.Abbreviations GlcNAc N-acetylglucosamine - TCA trichloroacetic acid - UDPGIcNAc uridine diphosphate-N-acetylglucosamine - DMSO dimethylsulfoxide