Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy

Valenciano, Susana; Lucas, J. Ramón De; Klei, Ida J. van der; Veenhuis, Marten; Laborda, F.

doi:10.1007/s002030050655

Cited by 40 publications

(31 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similar samples were also probed with anti-SKL antibodies that are expected to detect proteins targeted to peroxisomes and Woronin bodies (Fig. 7C) (16,32). The observed fluorescent pattern was consistent with localization to these organelles.…”

Section: Resultssupporting

confidence: 55%

“…In a previous study, the enzyme 6-aminopenicillanic acid acyltransferase, which catalyzes the final step of penicillin biosynthesis in Penicillium spp., was localized to a membrane-bound organelle, the microbody (25); the authors suggested that penicillin is synthesized in this organelle. In a separate study, prehelminthosporol, a phytotoxin produced by Bipolaris sorokiniana was localized in the Woronin body (1), which is thought to be derived from the peroxisome (32). Prehelminthosporol has been shown to disrupt plant plasma membranes and inhibit growth of gram-positive bacteria.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Function of Native OmtA In Vivo and Expression and Distribution of This Protein in Colonies of Aspergillus parasiticus

Lee¹,

Chiou²,

Linz

2002

Appl Environ Microbiol

View full text Add to dashboard Cite

The activities of two enzymes, a 168-kDa protein and a 40-kDa protein, OmtA, purified from the filamentous fungus Aspergillus parasiticus were reported to convert the aflatoxin pathway intermediate sterigmatocystin to O-methylsterigmatocystin in vitro. Our initial goal was to determine if OmtA is necessary and sufficient to catalyze this reaction in vivo and if this reaction is necessary for aflatoxin synthesis. We generated A. parasiticus omtA-null mutant LW1432 and a maltose binding protein-OmtA fusion protein expressed in Escherichia coli. Enzyme activity analysis of OmtA fusion protein in vitro confirmed the reported catalytic function of OmtA. Feeding studies conducted with LW1432 demonstrated a critical role for OmtA, and the reaction catalyzed by this enzyme in aflatoxin synthesis in vivo. Because of a close regulatory link between aflatoxin synthesis and asexual sporulation (conidiation), we hypothesized a spatial and temporal association between OmtA expression and conidiospore development. We developed a novel time-dependent colony fractionation protocol to analyze the accumulation and distribution of OmtA in fungal colonies grown on a solid medium that supports both toxin synthesis and conidiation. OmtA-specific polyclonal antibodies were purified by affinity chromatography using an LW1432 protein extract. OmtA was not detected in 24-h-old colonies but was detected in 48-h-old colonies using Western blot analysis; the protein accumulated in all fractions of a 72-h-old colony, including cells (0 to 24 h) in which little conidiophore development was observed. OmtA in older fractions of the colony (24 to 72 h) was partly degraded. Fluorescence-based immunohistochemical analysis conducted on thin sections of paraffin-embedded fungal cells from time-fractionated fungal colonies demonstrated that OmtA is evenly distributed among different cell types and is not concentrated in conidiophores. These data suggest that OmtA is present in newly formed fungal tissue and then is proteolytically cleaved as cells in that section of the colony age.

show abstract

Section: Resultssupporting

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Function of Native OmtA In Vivo and Expression and Distribution of This Protein in Colonies of Aspergillus parasiticus

Lee¹,

Chiou²,

Linz

2002

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Micrographs were overexposed (10,000 ms) for the green and red channels to highlight the lack of fluorescence or autofluorescence, except for ϩHxk2-GFP (1,400-ms green channel), ϩDsRed (3,200-ms red channel) and ϩHxk2-DsRed (1,400-ms red channel). (3,11,29,35,45,74) and in the basidiomycetes Fomitopsis palustris and Coprinus species (8,54,62). Thus, the peroxisomes of C. neoformans function somewhat similarly to those of S. cerevisiae, which are also not required for acetate metabolism (12) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Peroxisome Function Regulates Growth on Glucose in the Basidiomycete Fungus Cryptococcus neoformans

Idnurm¹,

Giles²,

Perfect

et al. 2007

Eukaryot Cell

View full text Add to dashboard Cite

The function of the peroxisomes was examined in the pathogenic basidiomycete Cryptococcus neoformans. Recent studies reveal the glyoxylate pathway is required for virulence of diverse microbial pathogens of plants and animals. One exception is C. neoformans, in which isocitrate lyase (encoded by ICL1) was previously shown not to be required for virulence, and here this was extended to exclude also a role for malate synthase (encoded by MLS1). The role of peroxisomes, in which the glyoxylate pathway enzymes are localized in many organisms, was examined by mutation of two genes (PEX1 and PEX6) encoding AAA (ATPases associated with various cellular activities)-type proteins required for peroxisome formation. The pex1 and pex6 deletion mutants were unable to localize the fluorescent DsRED-SKL protein to peroxisomal punctate structures, in contrast to wild-type cells. pex1 and pex6 single mutants and a pex1 pex6 double mutant exhibit identical phenotypes, including abolished growth on fatty acids but no growth difference on acetate. Because both icl1 and mls1 mutants are unable to grow on acetate as the sole carbon source, these findings demonstrate that the glyoxylate pathway can function efficiently outside the peroxisome in C. neoformans. The pex1 mutant exhibits wild-type virulence in a murine inhalation model and in an insect host, demonstrating that peroxisomes are not required for virulence under these conditions. An unusual phenotype of the pex1 and pex6 mutants was that they grew poorly with glucose as the carbon source, but nearly wild type with galactose, which suggested impaired hexokinase function and that C. neoformans peroxisomes might function analogously to the glycosomes of the trypanosomid parasites. Deletion of the hexokinase HXK2 gene reduced growth in the presence of glucose and suppressed the growth defect of the pex1 mutant on glucose. The hexokinase 2 protein of C. neoformans contains a predicted peroxisome targeting signal (type 2) motif; however, Hxk2 fused to fluorescent proteins was not localized to peroxisomes. Thus, we hypothesize that glucose or glycolytic metabolites are utilized in the peroxisome by an as yet unidentified enzyme or regulate a pathway required by the fungus in the absence of peroxisomes.

show abstract

“…First, it contains the peroxisome-targeting signal ARL. Second, our preliminary cell fractionation experiments using cell extracts from catA catB double mutant grown for 18 h showed that at least 20% of the total CatC activity is contained within the subcellular particle pellet, along with high activity levels of the peroxisomal marker isocitrate lyase (41) and the mitochondrial marker fumarase. Third, a catalase activity has been cytochemically localized in microbodies from young growing hyphae, and cosedimentation of catalase activity and peroxisome marker enzymes has also been shown in A. nidulans (42).…”

Section: Discussionmentioning

confidence: 99%

Multiple Catalase Genes Are Differentially Regulated in Aspergillus nidulans

2001

View full text Add to dashboard Cite

Detoxification of hydrogen peroxide is a fundamental aspect of the cellular antioxidant responses in which catalases play a major role. Two differentially regulated catalase genes, catA and catB, have been studied in Aspergillus nidulans. Here we have characterized a third catalase gene, designated catC, which predicts a 475-amino-acid polypeptide containing a peroxisome-targeting signal. With a molecular mass of 54 kDa, CatC shows high similarity to other small-subunit monofunctional catalases and is most closely related to catalases from other fungi, Archaea, and animals. In contrast, the CatA (ϳ84 kDa) and CatB (ϳ79 kDa) enzymes belong to a family of large-subunit catalases, constituting a unique fungal and bacterial group. The catC gene displayed a relatively constant pattern of expression, not being induced by oxidative or other types of stress. Targeted disruption of catC eliminated a constitutive catalase activity not detected previously in zymogram gels. However, a catalase activity detected in catA catB mutant strains during late stationary phase was still present in catC and catABC null mutants, thus demonstrating the presence of a fourth catalase, here named catalase D (CatD). Neither catC nor catABC triple mutants showed any developmental defect, and both mutants grew as well as wild-type strains in H 2 O 2 -generating substrates, such as fatty acids, and/or purines as the sole carbon and nitrogen sources, respectively. CatD activity was induced during late stationary phase by glucose starvation, high temperature, and, to a lesser extent, H 2 O 2 treatment. The existence of at least four differentially regulated catalases indicates a large and regulated capability for H 2 O 2 detoxification in filamentous fungi.Several studies indicate that reactive oxygen species play crucial roles in various aspects of cell physiology, such as cellular defense (45), life span (38), stress signaling (22), development (19), apoptosis (30), and pathology (33). The hydrogen peroxide formed during aerobic metabolism is capable of generating other reactive oxygen species, which can damage many cellular components (18). Catalases and peroxidases are the most important enzymatic systems used to degrade H 2 O 2 . There are three separate families of catalases: Mn-catalases, bifunctional catalase-peroxidases, and monofunctional, or "true," catalases. The last group is the one best characterized and corresponds to homotetrameric heme-containing enzymes present in eubacteria and eukaryotes and recently also found in the Archaea (34). Within this family of catalases, two clearly distinct classes can be recognized: the small-subunit (50-to 65-kDa) and the large-subunit (ϳ80-kDa) enzymes. The first class includes a large number of catalases from bacteria, plants, fungi, and animals. An increasing number of catalases of the second class have been identified in bacteria and filamentous fungi (5,8,13,15,(23)(24)(25)27, 37) but not in higher eukaryotes.The core sequence of the true catalases is composed of 360 to 390 amino acid resid...

show abstract

Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy

Cited by 40 publications

References 48 publications

Function of Native OmtA In Vivo and Expression and Distribution of This Protein in Colonies of Aspergillus parasiticus

Function of Native OmtA In Vivo and Expression and Distribution of This Protein in Colonies of Aspergillus parasiticus

Peroxisome Function Regulates Growth on Glucose in the Basidiomycete Fungus Cryptococcus neoformans

Multiple Catalase Genes Are Differentially Regulated in Aspergillus nidulans

Contact Info

Product

Resources

About