Abstract
Current studies in our laboratory demonstrate a functional link between vesicles, vacuoles and aflatoxin biosynthesis in the filamentous fungus, Aspergillus parasiticus. Under aflatoxin inducing conditions in liquid yeast-extract sucrose medium, A. parasiticus undergoes a shift from vacuole biogenesis to accumulation of an enhanced number of vesicles which exhibit significant heterogeneity in size and density. As a first step in conducting a detailed analysis of the role of these organelles in aflatoxin synthesis, we developed a novel method to purify the vesicle and vacuole fraction using protoplasts prepared from cells harvested during aflatoxin synthesis. The method includes the following steps: 1] preparation of protoplasts from mycelia grown for 36 h under aflatoxin inducing conditions; 2] release of vesicles and vacuoles from purified protoplasts in the presence of Triton X-100; and 3] fractionation of the vesicles and vacuoles using a "one-step high density cushion". The vesicle-vacuole fraction showed a 35 fold enrichment in alpha-mannosidase activity (vacuole marker) and non-detectable succinate dehydrogenase and lactate dehydrogenase activities (mitochondrial and cytoplasmic markers, respectively). Confocal laser scanning microscopy with the vacuole dyes MDY-64 and CMAC demonstrated that the fraction contained pure vesicles and vacuoles and was devoid of membranous debris. Transmission electron microscopy (TEM) confirmed that no mitochondria or unbroken protoplasts contaminated the purified fraction. The purified organelles exhibited significant size heterogeneity with a range of sizes similar to that observed in whole cells and protoplasts.