Functional Expression and Subcellular Localization of the Aflatoxin Pathway Enzyme Ver-1 Fused to Enhanced Green Fluorescent Protein

Abstract

Aflatoxin, a mycotoxin synthesized by Aspergillus spp., is among the most potent naturally occurring carcinogens known. Little is known about the subcellular organization of aflatoxin synthesis. Previously, we used transmission electron microscopy and immunogold labeling to demonstrate that the late aflatoxin enzyme OmtA localizes primarily to vacuoles in fungal cells on the substrate surface of colonies. In the present work, we monitored subcellular localization of Ver-1 in real time in living cells. Aspergillus parasiticus strain CS10-N2 was transformed with plasmid constructs that express enhanced green fluorescent protein (EGFP) fused to Ver-1. Analysis of transformants demonstrated that EGFP fused to Ver-1 at either the N or C terminus functionally complemented nonfunctional Ver-1 in recipient cells. Western blot analysis detected predominantly full-length Ver-1 fusion proteins in transformants. Confocal laser scanning microscopy demonstrated that Ver-1 fusion proteins localized in the cytoplasm and in the lumen of up to 80% of the vacuoles in hyphae grown for 48 h on solid media. Control EGFP (no Ver-1) expressed in transformants was observed in only 13% of the vacuoles at this time. These data support a model in which middle and late aflatoxin enzymes are synthesized in the cytoplasm and transported to vacuoles, where they participate in aflatoxin synthesis.Aflatoxins (AF) are toxic and carcinogenic secondary metabolites synthesized primarily by the filamentous fungi Aspergillus parasiticus and Aspergillus flavus when they grow on economically important food and feed crops, including peanuts, tree nuts, corn, and cottonseed (5,11,12,28,33). AF contamination of food and feed results in large economic losses and significant human and animal health risks (7,14). AF biosynthesis is a complex process that requires at least 17 enzyme activities encoded by 26 or more individual genes; these are clustered within a 70-kb region on one chromosome (42, 43). The biochemistry and molecular biology of AF synthesis have been studied intensively, but little is known about the subcellular organization of the AF pathway. Several enzyme activities involved in AFB 1 biosynthesis were detected in a microsomal fraction (4, 19, 41), suggesting membrane localization; other activities were found in the cytoplasm or loosely bound to membranes (4,19,35,41).Previously, we used immunogold labeling and transmission electron microscopy (TEM) to refine the localization analysis (21). Early (Nor-1), middle (Ver-1), and late (OmtA) AF biosynthetic pathway enzymes were observed primarily in the cytoplasm of fungal colonies grown 24 to 48 h on a solid, AFinducing medium. However, OmtA also was detected primarily in vacuoles in cells near the substrate surface of fungal colonies on solid media; we observed very little Nor-1 or Ver-1 in this location, and these proteins were not detected in vacuoles.ver-1 encodes a 28-kDa NADPH-dependent reductase involved in conversion of versicolorin A (VA) to demethylsterigmatocystin (15,17,37). Only one (...

A Possible Role for Exocytosis in Aflatoxin Export in Aspergillus parasiticus

“…Our previous data (3) strongly suggest that these vesicles are aflatoxisomes that carry aflatoxin and functional aflatoxin enzymes. In support of this idea, we previously observed that aflatoxin synthesis increases during a transition from active growth to stationary phase (24 to 36 h) (8). During this transition, the number of vesicles carrying the EGFP-tagged aflatoxin enzyme Ver-1 increased significantly (8) and these adhered to the inner surface of the cytoplasmic membrane.…”

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

“…There is evidence to suggest that many secondary metabolite biosynthetic pathways in fungi are restricted to specific organelles, as shown for cyclosporine, penicillin, and aflatoxin biosynthesis (51)(52)(53)(54)(55)(56). Recently specific synthesis vesicles, aflatoxisomes (57), were discovered in Aspergillus parasiticus.…”

A key role for vesicles in fungal secondary metabolism

“…The strains used in this study are listed in Table 1 : the model proposes that Nor-1 (early), Ver-1 (middle), and OmtA (late) are synthesized on free ribosomes in the cytoplasm, packaged into transport vesicles (6 -8), and transported to vacuoles via the cytoplasm-to-vacuole targeting (Cvt) pathway (6,8). In contrast Vbs localizes in the cytoplasm and in structures thought to be Golgi (29), suggesting it is transported to vacuoles via the classical secretory pathway; VBS is glycolsylated in support of this notion.…”

“…We previously observed that early (Nor-1), middle (Ver-1), and late (OmtA) aflatoxin enzymes localize in vesicles and vacuoles in A. parasiticus (6)(7)(8). To determine the functional role of these organelles in aflatoxin synthesis, a vesicle-vacuole fraction (V) was first isolated from A. parasiticus grown for 36 h in aflatoxin-inducing medium (YES) using a high-density sucrose cushion method.…”

“…1 and 2) and flavonoids (e.g., aurone) (reviewed in refs. 1 and 3) in plants and the nonribosomal peptide cyclosporin (4), the ␤-lactam antibiotic penicillin (5) (localization of ACVS is still controversial), and the polyketide aflatoxin (6)(7)(8) in fungi. However, the functional role of these compartments in secondary metabolism was unclear because these organelles potentially could be involved in synthesis, storage, protein turnover, transport, or export of the end product or biosynthetic enzymes.…”