title: Proteomic and biochemical evidence support a role for transport vesicles and endosomes in stress response and secondary metabolism in Aspergillus parasiticus.
abstract: Aflatoxin is among the most potent naturally occurring carcinogens known. Previous studies demonstrated that endosomes in the filamentous fungus Aspergillus parasiticus carry enzymes that catalyze the final two steps in aflatoxin synthesis, and these structures also play a role in aflatoxin storage and export. We hypothesized that endosomes house a complete and functional aflatoxin biosynthetic pathway. To address this hypothesis, we purified a cellular fraction containing endosomes, transport vesicles, and vacuoles (V fraction) from A. parasiticus grown under aflatoxin inducing and noninducing conditions. We also added (fed) aflatoxin pathway intermediates to V fraction to test the functional status of aflatoxin pathway enzymes. High throughput LC-MS/MS analysis of proteins in V fraction detected 8 aflatoxin enzymes with high reliability and 8 additional enzymes at lower reliability, suggesting that most aflatoxin pathway enzymes are present. Purified V fraction synthesized aflatoxin and addition of the pathway intermediate versicolorin A increased aflatoxin synthesis, confirming that middle and late aflatoxin enzymes in V fraction are functional. Of particular significance, proteomic and biochemical analysis strongly suggested that additional secondary metabolic pathways as well as proteins involved in response to heat, osmotic, and oxidative stress are housed in V fraction.
authors: Department of Food Science and Human Nutrition, Department of Microbiology and Molecular Genetics, 234B GM Trout Building, Michigan State University , East Lansing, Michigan 48824, United States. jlinz@msu.edu
authors: John E
authors: JE
authors: Linz
authors: Anindya
authors: A
authors: Chanda
authors: Sung-Yong
authors: SY
authors: Hong
authors: Douglas A
authors: DA
authors: Whitten
authors: Curtis
authors: C
authors: Wilkerson
authors: Ludmila V
authors: LV
authors: Roze
mesh_terms: D000348:Aflatoxins / Q000378:metabolism*; D001230:Aspergillus / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D002853:Chromatography, Liquid; D003470:Culture Media; D011992:Endosomes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D020543:Proteome / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D013312:Stress, Physiological; D053719:Tandem Mass Spectrometry; D022161:Transport Vesicles / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D014617:Vacuoles / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism
chemical_list: D000348:Aflatoxins; D001426:Bacterial Proteins; D003470:Culture Media; D020543:Proteome