A proteomic approach towards the identification of the matrix protein content of the two types of microbodies in Neurospora crassa.

Microbodies (peroxisomes) comprise a class of organelles with a similar biogenesis but remarkable biochemical heterogeneity. Here, we purified the two distinct microbody family members of filamentous fungi, glyoxysomes and Woronin bodies, from Neurospora crassa and analyzed their protein content by HPLC/ESI-MS/MS. In the purified Woronin bodies, we unambiguously identified only hexagonal 1 (HEX1), suggesting that the matrix is probably exclusively filled with the HEX1 hexagonal crystal. The proteomic analysis of highly purified glyoxysomes allowed the identification of 191 proteins. Among them were 16 proteins with a peroxisomal targeting signal type 1 (PTS1) and three with a PTS2. The collection also contained the previously described N. crassa glyoxysomal matrix proteins FOX2 and ICL1 that lack a typical PTS. Three PTS1 proteins were identified that likely represent the long sought glyoxysomal acyl-CoA dehydrogenases of filamentous fungi. Two of them were demonstrated by subcellular localization studies to be indeed glyoxysomal. Furthermore, two PTS proteins were identified that are suggested to be involved in the detoxification of nitroalkanes. Since the glyoxysomal localization was experimentally demonstrated for one of these enzymes, a new biochemical reaction is expected to be associated with microbody function.

Dynamin-like protein-dependent formation of Woronin bodies in Saccharomyces cerevisiae upon heterologous expression of a single protein.

Filamentous ascomycetes harbor Woronin bodies and glyoxysomes, two types of microbodies, within one cell at the same time. The dominant protein of the Neurospora crassa Woronin body, HEX1, forms a hexagonal core crystal via oligomerization and evidence has accumulated that Woronin bodies bud off from glyoxysomes. We analyzed whether HEX1 is sufficient to induce Woronin body formation upon heterologous expression in Saccharomyces cerevisiae, an organism devoid of this specialized organelle. In wild-type strain BY4742, initial import of HEX1 into existing peroxisomes enabled the formation of organelles with a hexagonal crystal. The observed structures mimicked the shape of genuine Woronin bodies, but exhibited a lower density and were significantly larger. Double-immunofluorescence analysis revealed that hexagonal HEX1 structures only occasionally co-localized with peroxisomal marker proteins, indicating that the Woronin-body-like structures are well separated from peroxisomes. In cells lacking Vps1p and Dnm1p, dynamin-like proteins required for the division of peroxisomes, the Woronin-body-like organelles remained attached to peroxisomes. The data indicate that Woronin bodies emerge after the formation of a HEX1 core crystal within peroxisomes followed by Vps1p- and Dnm1p-mediated fission.

Identification of PEX33, a novel component of the peroxisomal docking complex in the filamentous fungus Neurospora crassa.

The docking complex of peroxisomal matrix protein import is composed of PEX13 and PEX14 in all species analyzed so far, whereas only yeast appears to possess an additional component, PEX17. In this report we isolated PEX14 complexes of Neurospora crassa. Among the complex constituents, one protein designated as PEX33 possessed homology to PEX14 but only in a short N-terminal domain. The PEX14/PEX33 interaction was verified by means of two-hybrid analysis. Moreover, PEX33 was shown to interact with itself and the PTS1-receptor PEX5. Localization studies demonstrated that PEX33 constitutes a glyoxysomal protein. Growth tests of the pex33 deletion strain revealed a defect of this strain in the biogenesis of glyoxysomes and Woronin bodies. As the function of PEX33 was not redundant to that of PEX14, it is a genuine novel peroxin. Based on our experimental data, the function of PEX33 seems to resemble that of yeast PEX17 despite clear structural differences.

The peroxin PEX14 of Neurospora crassa is essential for the biogenesis of both glyoxysomes and Woronin bodies.

In the filamentous fungus Neurospora crassa, glyoxysomes and Woronin bodies coexist in the same cell. Because several glyoxysomal matrix proteins and also HEX1, the dominant protein of Woronin bodies, possess typical peroxisomal targeting signals, the question arises as to how protein targeting to these distinct yet related types of microbodies is achieved. Here we analyzed the function of the Neurospora ortholog of PEX14, an essential component of the peroxisomal import machinery. PEX14 interacted with both targeting signal receptors and was localized to glyoxysomes but was virtually absent from Woronin bodies. Nonetheless, a pex14Delta mutant not only failed to grow on fatty acids because of a defect in glyoxysomal beta-oxidation but also suffered from cytoplasmic bleeding, indicative of a defect in Woronin body-dependent septal pore plugging. Inspection of pex14Delta mutant hyphae by fluorescence and electron microscopy indeed revealed the absence of Woronin bodies. When these cells were subjected to subcellular fractionation, HEX1 was completely mislocalized to the cytosol. Expression of GFP-HEX1 in wild-type mycelia caused the staining of Woronin bodies and also of glyoxysomes in a targeting signal-dependent manner. Our data support the view that Woronin bodies emerge from glyoxysomes through import of HEX1 and subsequent fission.

The WW domain protein PRO40 is required for fungal fertility and associates with Woronin bodies.

Fruiting body formation in ascomycetes is a highly complex process that is under polygenic control and is a fundamental part of the fungal sexual life cycle. However, the molecular determinants regulating this cellular process are largely unknown. Here we show that the sterile pro40 mutant is defective in a 120-kDa WW domain protein that plays a pivotal role in fruiting body maturation of the homothallic ascomycete Sordaria macrospora. Although WW domains occur in many eukaryotic proteins, homologs of PRO40 are present only in filamentous ascomycetes. Complementation analysis with different pro40 mutant strains, using full-sized or truncated versions of the wild-type pro40 gene, revealed that the C terminus of PRO40 is crucial for restoring the fertile phenotype. Using differential centrifugation and protease protection assays, we determined that a PRO40-FLAG fusion protein is located within organelles. Further microscopic investigations of fusion proteins with DsRed or green fluorescent protein polypeptides showed a colocalization of PRO40 with HEX-1, a Woronin body-specific protein. However, the integrity of Woronin bodies is not affected in mutant strains of S. macrospora and Neurospora crassa, as shown by fluorescence microscopy, sedimentation, and immunoblot analyses. We discuss the function of PRO40 in fruiting body formation.

A eukaryote without catalase-containing microbodies: Neurospora crassa exhibits a unique cellular distribution of its four catalases.

Microbodies usually house catalase to decompose hydrogen peroxide generated within the organelle by the action of various oxidases. Here we have analyzed whether peroxisomes (i.e., catalase-containing microbodies) exist in Neurospora crassa. Three distinct catalase isoforms were identified by native catalase activity gels under various peroxisome-inducing conditions. Subcellular fractionation by density gradient centrifugation revealed that most of the spectrophotometrically measured activity was present in the light upper fractions, with an additional small peak coinciding with the peak fractions of HEX-1, the marker protein for Woronin bodies, a compartment related to the microbody family. However, neither in-gel assays nor monospecific antibodies generated against the three purified catalases detected the enzymes in any dense organellar fraction. Furthermore, staining of an N. crassa wild-type strain with 3,3'-diaminobenzidine and H(2)O(2) did not lead to catalase-dependent reaction products within microbodies. Nonetheless, N. crassa does possess a gene (cat-4) whose product is most similar to the peroxisomal type of monofunctional catalases. This novel protein indeed exhibited catalase activity, but was not localized to microbodies either. We conclude that N. crassa lacks catalase-containing peroxisomes, a characteristic that is probably restricted to a few filamentous fungi that produce little hydrogen peroxide within microbodies.

Comparative sequence analysis of Sordaria macrospora and Neurospora crassa as a means to improve genome annotation.

One of the most challenging parts of large scale sequencing projects is the identification of functional elements encoded in a genome. Recently, studies of genomes of up to six different Saccharomyces species have demonstrated that a comparative analysis of genome sequences from closely related species is a powerful approach to identify open reading frames and other functional regions within genomes [Science 301 (2003) 71, Nature 423 (2003) 241]. Here, we present a comparison of selected sequences from Sordaria macrospora to their corresponding Neurospora crassa orthologous regions. Our analysis indicates that due to the high degree of sequence similarity and conservation of overall genomic organization, S. macrospora sequence information can be used to simplify the annotation of the N. crassa genome.