The role of the endomembrane system during gametogenesis in Allomyces macrogynus

Abstract

Allomyces macrogynus produces uninucleate, motile gametes by the rearrangement and cleavage of the multinucleate cytoplasm of gametangia. In other aquatic fungi, a Golgi complex made up of stacked cisternae is the source of new membrane and glycoproteins formed during cytoplasmic cleavage. Allomyces does not have stacks of Golgi cisternae but instead has individual smooth endomembrane elements, termed Golgi equivalents. The objective of this study was to use an ultrastructural, pharmacological, and biochemical approach to determine the origin of cleavage furrow membrane during gametogenesis. The microdroplet culture method developed for this study gave a 1.7 to 2.4-fold higher degree of synchrony during gametogenesis. Using this method, it was possible to construct a precise timing sequence for gamete differentiation. Straining with ZnI-OsO₄ impregnation demonstrated that the endoplasmic reticulum was the origin of the nuclear cap membrane but neither the cleavage furrows nor the membrane elements which gave rise to them strained with ZnI-OsO₄. Since acidic compartments, such as trans Golgi do not stain by this method it appeared likely that cleavage furrows might be derived from Golgi equivalents. To further test the hypothesis that Golgi equivalents were involved in cleavage, induced gametangia were treated with monensin, an ionophore which specifically affects trans Golgi cisternae. Gametangia treated with monensin did not complete cytoplasmic cleavage but, instead, released multinucleate gametes. Although swollen vacuoles were present, other gametogenic events including nuclear cap membrane formation occurred normally. Fractionation of subcellular components on sucrose density gradients demonstrated that Allomyces had both endoplasmic reticulum and Golgi equivalents with buoyant densities and marker enzyme profiles typical for these components in cells with stacked Golgi cisternae. For the first time, it was possible to demonstrate developmentally-regulated glycoproteins in vegetative hyphae and gametangia of Allomyces. Some of these glycoproteins appear in Golgi fractions, implying that the Golgi equivalents might be involved in glycosylation. In summary, Allomyces has a Golgi complex composed of individual or, perhaps, stacks of two Golgi equivalents. Although the endoplasmic reticulum was the source of the nuclear cap membrane, Golgi equivalents were the origin of cleavage furrow membranes formed during gametogenesis.