Yeast Cells Of Mucor Rouxii Research Articles

Protein composition of purified chitosomes of Mucor rouxii

Different centrifugation conditions were compared for purification of chitosomes (microvesicles containing chitin synthetase). Isopycnic centrifugation of crude chitosome samples from yeast cells of Mucor 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 of M. 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.

An efficient preparative isopycnic flotation method for the isolation of chitosomes

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 Rav). 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.

Sterol composition of chitosomes from yeast cells of Mucor rouxii: Comparison with whole cells

The free sterol composition of chitosomes and whole yeast cells of Mucor rouxii was determined by gas-liquid chromatography (GLC). The results obtained showed that, in contrast to whole cells, chitosomes are particularly rich in ergosterol: 87% vs. 29% of total free sterols. Three other sterols were present in varying amounts in both chitosomes and whole cells. These were identified by GLC as cholesterol, stigmasterol and sitosterol. These results indicate that the yeast cells preferentially accumulate ergosterol in chitosomes providing these microvesicles with a unique composition that may be important for the structure of the organelle.

Sterol composition of chitosomes from yeast cells ofMucor rouxii: Comparison with whole cells

The free sterol composition of chitosomes and whole yeast cells of Mucor rouxii was determined by gas-liquid chromatography (GLC). The results obtained showed that, in contrast to whole cells, chitosomes are particularly rich in ergosterol: 87% vs. 29% of total free sterols. Three other sterols were present in varying amounts in both chitosomes and whole cells. These were identified by GLC as cholesterol, stigmasterol and sitosterol. These results indicate that the yeast cells preferentially accumulate ergosterol in chitosomes providing these microvesicles with a unique composition that may be important for the structure of the organelle.

Sedimentation properties of chitosomes fromMucor rouxii

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.

Effects of amphotericin B, nystatin, and other polyene antibiotics on chitin synthase.

The effects of amphotericin B (AmB), nystatin, filipin, and pimaricin were tested chitin synthase (EC 2.4.1.16) (chitosomes from yeast cells of Mucor rouxii). AmB and nystatin inhibited the enzyme at concentrations greater than or equal to 10 micrograms/ml, filipin was weakly inhibitory, and pimaricin had no effect. The inhibition of chitin synthase by AmB appears to be noncompetitive, with a Ki value of about 0.13 mM. the effect of nystatin was more complex and included a sharp stimulation of chitin synthase activity at approximately 50 micrograms/ml. Our findings suggest the existence of binding sites (sterols?) on the chitosome that are selective for certain polyenes and that play a role in the operation of chitin synthase. Because the minimal growth inhibitory concentrations of AmB or nystatin are lower than the concentrations that inhibit chitin synthase in vitro, the possibility of chitosomal chitin synthase being a primary target for the antifungal action of these polyenes seems unlikely.

Isolation of chitosomes from taxonomically diverse fungi and synthesis of chitin microfibrils in Vitro

Small spheroidal structures (mostly 40–70 nm in diameter) similar in shape, size, and chitin-synthesizing ability to the chitosomes described earlier for yeast cells of Mucor rouxii were isolated from four other fungi: Allomyces macrogynus (Emerson) Emerson and Wilson, Saccharomyces cerevisiae Meyen ex Hansen, Neurospora crassa Shear and Dodge, and Agaricus bisporus (Lange) Imbach. Chitosomes were also found in the mycelial form of M. rouxii. Evidence of zymogenicity of chitin synthetase was found in crude preparations from all five fungal genera in that protease treatment caused an increase in chitin synthetase activity. There were, however, specific differences among the fungi in the response of the zymogen to acid or neutral proteases and in the retention of zymogenicity during chitosome isolation. Upon addition of substrate (uridine diphosphate N-acetyl-D-glucosamine) and activating protease (if needed), the isolated chitosome samples produced chitin microfibrils. Both “fibroid” coils and extended microfibrils were seen. The isolation of functional chitosomes from five widely diverse genera, representing five major fungal groups (Zygomycetes, Chytridiomycetes, Hemiascomycetes, Ascomycetes, and Basidiomycetes) and three distinct developmental forms (hyphase, yeast cells, and basidiocarp), suggests that the chitosomes are a ubiquitous component of the cytoplasmic mechanism that conveys chitin synthetase to the sites of microfibril assembly at the surface of fungal cells.

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.

Carbon Dioxide Fixation by Yeast Cells of Mucor rouxii

Carbon Dioxide Fixation by Yeast Cells of Mucor rouxii

Carbon Dioxide Fixation by Yeast Cells ofMucor Rouxii

Carbon Dioxide Fixation by Yeast Cells of<i>Mucor Rouxii</i>

Microfibril assembly by granules of chitin synthetase.

Purified preparations of chitin synthetase (EC 2.4.1.16; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase), capable of forming microfibrils in vitro, were isolated from yeast cells of Mucor rouxii. Chitin synthetase was obtained either by substrate-induced liberation of bound enzyme (54,000 x g pellet) or by isolation of unbound enzyme present in the 54,000 x g supernatant of a cell-free extract. Both preparations contained ellipsoidal granules from about 350 to 1000 A diameter. Many granules exhibited a marked depression. No typical unit membrane profiles appeared in thin sections of glutaraldehyde/OsO4-fixed samples. Upon incubation with substrate and activators, chitin microfibrils were produced. The microfibrils were often found intimately associated with granules. The most common configurations were: a microfibril with a granule at one end, or two microfibrils "arising" from the same granule. These findings lend support to the granule hypothesis for the elaboration of cell wall microfibrils by end-synthesis.