Subcellular localization of aflatoxin enzymes and aflatoxin in Aspergillus parasiticus

Aflatoxin biosynthesis initiates as a response to environmental changes, involves a tightly regulated expression of a 75kb gene cluster resulting at least 17 enzyme activities and finally ends with the secretion of end products outside the cell. It is one of the most characterized eukaryotic secondary metabolic pathways and hence serves as a useful model to understand secondary metabolism in fungi and plants. Understanding the spatial distribution of proteins and biosynthetic products in Aspergillus parasiticus during aflatoxin synthesis helps us to understand fungal and plant secondary metabolism in general at a cellular level. Prior to our current work, the intracellular site of aflatoxin synthesis was unknown although our previous subcellular studies demonstrated localization of early, middle and late aflatoxin enzymes in vesicles and vacuoles during aflatoxin synthesis at peak levels.;Under aflatoxin inducing conditions in liquid yeast-extract sucrose medium, A. parasiticus generates vesicles and vacuoles which exhibit significant heterogeneity in size (20nm to 5mum) and density. The hypothesis tested in this study is vesicles and vacuoles are the intracellular site(s) for aflatoxin biosynthesis. Two independent approaches were used to test the hypothesis.;As a first step in conducting a detailed analysis of the role of these organelles in aflatoxin synthesis, a novel "high density sucrose cushion" method was developed to purify a vesicle-vacuole fraction using protoplasts prepared from cells harvested during aflatoxin synthesis. Western blot analysis demonstrated the presence of three aflatoxin enzymes (Ver-1, Vbs and OmtA) in this fraction. Vesicles and vacuoles also were observed to represent a primary site to sequester aflatoxin. This purified fraction converted sterigmatocystin, a late intermediate in aflatoxin biosynthesis, to aflatoxin B1 showing that the last two steps of aflatoxin biosynthesis occur in or on vesicles and vacuoles.;Second, to clarify which compartments (vesicles or vacuoles) were functionally involved aflatoxin biosynthesis vesicle-vacuole fusion (a late step in vacuole biogenesis in eukaryotes) was interrupted by application of Sortin3 (a low mass bioactive compound that interferes with protein trafficking to the vacuole and produces a vps16 phenotype in yeast) and by disruption of the Rab7/Ypt7 GTPase homologue, vb1. Both treatments resulted in accumulation of vesicles and significantly higher levels of aflatoxins in the medium compared to wild-type (ELISA). This increase in aflatoxin was not due to upregulation in aflatoxin gene expression (RT-PCR) but was due to significantly higher accumulation of functional enzymes (Western blot analysis) caused by elevated accumulation of vesicles. These data provide the first evidence that manipulation of the vesicle transport machinery in Aspergillus affects secondary metabolism. We hypothesize that vesicles represent the intracellular site for the late steps of aflatoxin biosynthesis and aflatoxin export, whereas the vacuole is primarily responsible for aflatoxin storage and aflatoxin enzyme turnover.