Structure and transformation of chitin synthetase particles (chitosomes) during microfibril synthesis in vitro.

Abstract

The fine structure of isolated chitin synthetase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-,B-acetamidodeoxyglucosyltransferase; EC 2.4.1.16) particles (chitosomes) from Mucor rouxii and the elaboration of chitin microfibrils were studied by electron microscopy. Chitosomes are spheroidal, but often polymorphic, structures, mostly 40-70 nm in diameter. Their appearance after negative staining varies. Some reveal internal granular structure enclosed by a shell measuring 6-12 nm thick; others do not show internal structure but have a pronounced depression of the external surface. In thin sections, isolated chitosomes appear as microvesicular structures with a tripartite shell 6.5-7.0 nm thick. Morphologically similar structures can be seen in intact cells of M. rouxii. Isolated chitosomes undergo a seemingly irreversible series of transformations when substrate and activators are added. The internal structure changes, and a coiled microfibril (fibroid) appears inside the chitosome. The shell of the chitosome is opened or shed, and an extended microfibril arises from the fibroid particle. During prolonged incubation, the fibroid coils become less common and extended microfibrils appear thicker. We regard the chitosome as the cytoplasmic container and conveyor of chitin synthetase en route to its destination at the cell surface. Isolated chitosomes are well suited for integrated ultrastructural-biochemical studies of microfibril biogenesis in vitro. Chitin microfibrils can be assembled in vitro by chitin synthetase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-j3-acetamidodeoxyglucosyltransferase; EC 2.4.1.16) preparations isolated from the yeast form bf the fungus Mucor rouxii (1, 2). Electron microscopy of shadow cast specimens showed that the fibrils were formed from particles ("granules") smaller than 100 nm in diameter (2). These particles, which we now call chitosomes, contain a chitin synthetase complex capable of forming a microfibril by collective synthesis of the polysaccharide chains. The biochemical properties of chitosomal chitin synthetase will be reported elsewhere. An understanding of the underlying molecular mechanism of microfibril biogenesis requires knowledge of the detailed fine structure of the enzyme complex and the way in which microfibrils are assembled. Here, we show the first evidence of the morphological complexity of chitosomes as well as the striking transformations they undergo during the course of microfibril elaboration.MATERIALS AND METHODS Isolation and Purification of Chitin Synthetase. Mucor rouxii strain IM-80 was grown in liquid medium (0.3% yeast extract, 1% peptone, 2% glucose, pH 4.5) at 28°for 13 hr in a N2:CO2 (30%:70%) atmosphere (2). Cells were harvested, mixed with 50 mM KH2PO4-NaOH buffer, pH 6.5, and 10 mM MgCl2, and broken in a Braun model MSK cell homogenizer (2). All subsequent manipulations were done in this buffer.After cell walls were removed at 1000 X g for 5 min, the cellfree extracts were centrifuged at 54,000 X g (Rav) for 45 min.The supernatant was ...

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

“…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…”

Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases

“…In fungal systems, substantial data have accumulated indicating that chitin synthase activity of at least one chitin synthase isoform (CHS3p) is associated with specialized intracellular microvesicles, known as chitosomes, which exhibit a special lipid and protein composition (Bracker et al, 1976;Hernandez et al, 1981;Florez-Martinez et al, 1990). Electron microscopy has revealed that, in the presence of UDP-GlcNAc and activators, purified chitosomes synthesize microfibrils that crystallize in the lumen of the vesicles (Bracker et al, 1976).…”

Polar Localizing Class V Myosin Chitin Synthases Are Essential during Early Plant Infection in the Plant Pathogenic FungusUstilago maydis

“…The synthesis of chitin is mediated by membrane-bound chitin synthases (CHSs) that locate to specialized transport vesicles, the chitosomes (Bracker et al, 1976;Leal-Morales et al, 1994;Sietsma et al, 1996) or to the plasma membrane (Duran et al, 1979;Leal-Morales et al, 1988;Martinez and Gozalbo, 1994). The yeast Saccharomyces cerevisiae contains three CHSs that have cell cycle-specific functions in septum formation and during budding (Cabib et al, 2001).…”