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Updated: Jun 24, 2026
Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation
Published on: August 21, 2017
Purification of a vesicle-vacuole fraction functionally linked to aflatoxin synthesis in Aspergillus parasiticus
Anindya Chanda1, Ludmila V Roze, Alicia Pastor
1Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824, USA.
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.
Insights
Researchers linked vesicles and vacuoles to aflatoxin biosynthesis in Aspergillus parasiticus. A new method purifies these organelles, crucial for understanding fungal toxin production.
Area of Science:
- Mycology
- Cell Biology
- Biochemistry
Background:
- A functional link between vesicles, vacuoles, and aflatoxin biosynthesis exists in Aspergillus parasiticus.
- A. parasiticus shifts from vacuole biogenesis to vesicle accumulation under aflatoxin-inducing conditions.
Purpose of the Study:
- To develop a novel method for purifying vesicle and vacuole fractions from A. parasiticus.
- To facilitate detailed analysis of the role of these organelles in aflatoxin synthesis.
Main Methods:
- Protoplast preparation from A. parasiticus mycelia grown under aflatoxin-inducing conditions.
- Release of vesicles and vacuoles using Triton X-100.
- Fractionation using a one-step high-density cushion, followed by microscopy and enzyme assays.
Main Results:
- A 35-fold enrichment of alpha-mannosidase activity (vacuole marker) was observed.
- Mitochondrial and cytoplasmic markers were undetectable, indicating purity.
- Confocal microscopy and TEM confirmed the absence of contaminants and the presence of heterogeneous vesicles and vacuoles.
Conclusions:
- A robust method for purifying vesicle and vacuole fractions from A. parasiticus was established.
- The purified fraction is suitable for studying the role of these organelles in aflatoxin biosynthesis.
