Microfibril assembly by granules of chitin synthetase.

Ruíz-Herrera, José; Sing, V.O.; Woude, William J. Van Der; Bartnicki-García, S.

doi:10.1073/pnas.72.7.2706

Cited by 90 publications

(24 citation statements)

References 15 publications

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“…The presence of such an inactive enzyme has also been demonstrated in 54000g supernatants from cell-free extracts from yeast cells of Mucor rouxii, where it was associated with identifiable particles called chitosomes (Ruiz-Herrera et al, 1975;Bracker et al, 1976). The role of these chitosomes in chitin synthesis has not yet been elucidated but it was suggested that they are extruded through the plasma membrane…”

Section: Discussionmentioning

confidence: 99%

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Localization of Chitin Synthase Activity in Subcellular Fractions of Schizophyllum commune Protoplasts

Vermeulen

Raeven

Wessels

1979

Journal of General Microbiology

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Plasma membranes of Schizophyllum commune were stabilized against fragmentation by coating protoplasts with concanavalin A (Con A). A uniform distribution of Con A over the membrane was demonstrated cytochemically. After lysis of the protoplasts in the presence of an inhibitor of proteolysis, plasma membranes were purified and, together with other subcellular fractions, assayed for chitin synthase activity. About 50% of the chitin synthase activity was associated with the plasma membranes and largely occurred in an active form. About 30% of the chitin synthase, in an inactive form, was recovered in fractions sedimenting at 40000 to 300000 g. The product of the plasma membrane-bound enzyme was shown to be a-chitin occurring as microfibrils (about 6 nm wide). The chitin synthase could not be detached from the plasma membranes by incubation with substrate and activator at 0 "C and gradient centrifugation showed that the synthesized chitin remained associated with the plasma membranes.

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Section: Discussionmentioning

confidence: 99%

“…by incubating these preparations with the substrate uridine-5'-diphospho-N-acetyl-~-glucosamine (UDP-GlcNAc) and the activator N-acetylglucosamine (GlcNAc) at 0 "C (Ruiz-Herrera & Bartnicki-Garcia, 1974 ;Ruiz-Herrera et al, 1975).…”

Section: A V E R M E U L E N M B J M R a E V E N A N D J Gmentioning

confidence: 99%

Localization of Chitin Synthase Activity in Subcellular Fractions of Schizophyllum commune Protoplasts

Vermeulen

Raeven

Wessels

1979

Journal of General Microbiology

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“…In N. crassa, the phosphatase PP1 (NCU00043), with a potential role in dephosphorylation of CHS-4 and another protein of potentially great interest, the serine protease p2, were identified in co-immunoprecipitations of CHS-4 and CHS-5. It was previously described that CHSs are zymogen proteins, which need to be activated proteolytically (Cabib and Farkas, 1971;Duran and Cabib, 1978;Leal-Morales et al, 1988;McMurrough and BartnickiGarcía, 1971;Ruiz-Herrera et al, 1975). Previous studies in N. crassa have shown that the total activity of CHSs requires trypsin as proteolytic activator (Arroyo-Begovich and Ruiz-Herrera, 1979).…”

Section: Discussionmentioning

confidence: 99%

Dissecting the function of the different chitin synthases in vegetative growth and sexual development in Neurospora crassa

Fajardo-Somera¹,

Jöhnk²,

Bayram³

et al. 2015

Fungal Genetics and Biology

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a b s t r a c tChitin, one of the most important carbohydrates of the fungal cell wall, is synthesized by chitin synthases (CHS). Seven sequences encoding CHSs have been identified in the genome of Neurospora crassa. Previously, CHS-1, -3 and -6 were found at the Spitzenkörper (Spk) core and developing septa. We investigated the functional importance of each CHS in growth and development of N. crassa. The cellular distribution of each CHS tagged with fluorescent proteins and the impact of corresponding gene deletions on vegetative growth and sexual development were compared. CHS-2, -4, -5 and -7 were also found at the core of the Spk and in forming septa in vegetative hyphae. As the septum ring developed, CHS-2-GFP remained at the growing edge of the septum until it localized around the septal pore. In addition, all CHSs were located in cross-walls of conidiophores. A partial co-localization of CHS-1-m and CHS-5-GFP or CHS-2-GFP occurred in the Spk and septa. Analyses of deletion mutants suggested that CHS-6 has a role primarily in hyphal extension and ascospore formation, CHS-5 in aerial hyphae, conidia and ascospore formation, CHS-3 in perithecia development and CHS-7 in all of the aforementioned. We show that chs-7/ csmB fulfills a sexual function and chs-6/chsG fulfills a vegetative growth function in N. crassa but not in Aspergillus nidulans, whereas vice versa chs-2/chsA fulfills a sexual function in A. nidulans but not in N. crassa. This suggests that different classes of CHSs can fulfill distinct developmental functions in various fungi. Immunoprecipitation followed by mass spectrometry of CHS-1-GFP, CHS-4-GFP and CHS-5-GFP identified distinct putative interacting proteins for each CHS. Collectively, our results suggest that there are distinct populations of chitosomes, each carrying specific CHSs, with particular roles during different developmental stages.

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“…The duration of the preincubation of chitin synthase with Rennilase and polyene prior to the addition of the substrate was 20 min.The reaction time was 1 hr and the temperature was 220C. The radioactive chitin formed was determined by a filtration method (5,16). The reaction rate (in the absence of polyene) was constant for at least 90 min, even with the most active preparations.…”

Section: Methodsmentioning

confidence: 99%

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

Rast¹,

Bartnicki-García²

1981

Proc. Natl. Acad. Sci. U.S.A.

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The effects of amphotericin B (AmB), nystatin, filipin, and pimaricin were tested on purified chitin synthase (EC 2.4.1.16) (chitosomes from yeast cells of Mucor rouxii). AmB and nystatin inhibited the enzyme at concentrations 210 #g/ml, filiin was weakly inhibitory, and pimaricin had no effect. The inhibition of chitin synthase by AmB appears to be noncompetitive, with a K; value of about 0.13 mM. The effect of nystatin was more complex and included a sharp stimulation of chitin synthase activity at "'50 jug/ml. Our Chitosomes are microvesicular organelles that can synthesize chitin fibrils in vitro (1-4). Chitosomes have been isolated from representatives of all major groups of the chitinous fungi (5). Digitonin, a well known sterol-complexing agent (6, 7), has profound effects on chitosomes. Depending on concentration, this saponin can stimulate or inhibit the chitin synthase activity (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-,B-acetamidodeoxy-D-glucosyltransferase, EC 2.4.1.16) of chitosomes from Mucor rouxii and cause their 'dissociation into subunits (8)."Solubilization" (9, 10) and inhibition (9) of chitin synthase by digitonin have also been noted for other fungi.The interaction of digitonin with chitosomal chitin synthase prompted us to examine the effect of polyene antibiotics on chitosomes because these agents have, like digitonin, a marked sterol-binding capacity (11-15). MATERIALS AND METHODSGrowth of the yeast form of Mucor rouxii, IM-80 (American Type Culture Collection 24905), and preparation of purified chitosomes were done as described (5). Chitosomes were kept at 4°C and used within 24 hr.Chitin Synthase Assay. The standard incubation mixture contained, in a total volume of 125 ,ul, enzyme preparation (50 p1), polyene solution (25 ,ul), and the following final concentrations of reagents: Rennilase (a crude acid protease; 1 mg/ ml), UDP-['4CJGlcNAc (0.4 mM; 22,500 dpm), GIcNAc (20 mM), and ATP (0.2 mM). All solutions were prepared in 0.05 M KH2POjNaOH buffer containing 0.01 M MgCl2, pH 6.5 (hereafter referred to as "buffer"). The duration of the preincubation of chitin synthase with Rennilase and polyene prior to the addition of the substrate was 20 min.The reaction time was 1 hr and the temperature was 220C. The radioactive chitin formed was determined by a filtration method (5, 16). The reaction rate (in the absence of polyene) was constant for at least 90 min, even with the most active preparations.Growth Inhibition Tests. Sporangiospores of M. rouxii, prepared as described (17), were inoculated into 125-ml Erlenmeyer flasks with 25 ml of YPG medium (18) to a final density of 104 spores per ml. The antibiotics were dissolved in either dimethyl sulfoxide or N,N-dimethylformamide, and 250 Al of such solutions was added to each culture. These two solvents caused slight growth inhibition of M. rouxii. Growth inhibition (percent) was calculated relative to control cultures with dimethyl sulfoxide or dimethylformamide. The flasks were incubated aerobically on a shaker at 30°C in the da...

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Microfibril assembly by granules of chitin synthetase.

Cited by 90 publications

References 15 publications

Localization of Chitin Synthase Activity in Subcellular Fractions of Schizophyllum commune Protoplasts

Localization of Chitin Synthase Activity in Subcellular Fractions of Schizophyllum commune Protoplasts

Dissecting the function of the different chitin synthases in vegetative growth and sexual development in Neurospora crassa

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

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