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.