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Receptor for Activated C Kinase-1 (RACK1) is a multifunctional eukaryotic scaffolding protein with a seven WD repeat structure. Among their many cellular roles, RACK1 homologs have been shown to serve as alternative Gβ subunits during heterotrimeric G protein signaling in many systems. We investigated genetic interactions between the RACK1 homolog cpc-2, the previously characterized Gβ subunit gnb-1 and other G protein signaling components in the multicellular filamentous fungus Neurospora crassa. Results from cell fractionation studies and from fluorescent microscopy of a strain expressing a CPC-2-GFP fusion protein revealed that CPC-2 is a cytoplasmic protein. Genetic epistasis experiments between cpc-2, the three Gα genes (gna-1, gna-2 and gna-3) and gnb-1 demonstrated that cpc-2 is epistatic to gna-2 with regards to basal hyphae growth rate and aerial hyphae height, while deletion of cpc-2 mitigates the increased macroconidiation on solid medium observed in Δgnb-1 mutants. Δcpc-2 mutants inappropriately produce conidiophores during growth in submerged culture and mutational activation of gna-3 alleviates this defect. Δcpc-2 mutants are female-sterile and fertility could not be restored by mutational activation of any of the three Gα genes. With the exception of macroconidiation on solid medium, double mutants lacking cpc-2 and gnb-1 exhibited more severe defects for all phenotypic traits, supporting a largely synergistic relationship between GNB-1 and CPC-2 in N. crassa., Competing Interests: The authors have declared that no competing interests exist.

Aspergillus fumigatus is the most common pathogenic mold infecting humans and a significant cause of morbidity and mortality in immunocompromised patients. In invasive pulmonary aspergillosis, A. fumigatus spores are inhaled into the lungs, undergoing germination and invasive hyphal growth. The fungus occludes and disrupts the blood vessels, leading to hypoxia and eventual tissue necrosis. The ability of this mold to adapt to hypoxia is regulated in part by the sterol regulatory element binding protein (SREBP) SrbA and the DscA to DscD Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. Loss of the genes encoding these proteins results in avirulence. To identify novel regulators of hypoxia sensing, we screened the Neurospora crassa gene deletion library under hypoxia and identified a novel rhomboid family protease essential for hypoxic growth. Deletion of the A. fumigatus rhomboid homolog rbdA resulted in an inability to grow under hypoxia, hypersensitivity to CoCl2, nikkomycin Z, fluconazole, and ferrozine, abnormal swollen tip morphology, and transcriptional dysregulation-accurately phenocopying deletion of srbA. In vivo, rbdA deletion resulted in increased sensitivity to phagocytic killing, a reduced inflammatory Th1 and Th17 response, and strongly attenuated virulence. Phenotypic rescue of the ΔrbdA mutant was achieved by expression and nuclear localization of the N terminus of SrbA, including its HLH domain, further indicating that RbdA and SrbA act in the same signaling pathway. In summary, we have identified RbdA, a novel putative rhomboid family protease in A. fumigatus that mediates hypoxia adaptation and fungal virulence and that is likely linked to SrbA cleavage and activation., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

A gene for ribosomal protein P0 that belongs to the family of ribosomal P proteins was isolated from a Neurospora crassa cDNA library, using polyclonal antibodies against recombinant P0 protein from Saccharomyces cerevisiae. This is the first gene for ribosomal P0 protein to be cloned from filamentous fungi. The derived P0 protein sequence has a strong homology to other eukaryotic P0 proteins; yet, there is a notable alteration in the conservative C-terminal region, placing this protein among the unique sequences from protozoan parasites.

Genes encoding subunits of complex I (EC 1.6.5.3) of the mitochondrial respiratory chain vary in their locations between the mitochondrial and nuclear genomes in different organisms, whereas genes for a homologous multisubunit complex in chloroplasts have to date only been found on the plastid genome. In potato (Solanum tuberosum L.), the gene coding for the mitochondrial 76 kDa iron-sulphur protein is identified in the nuclear genome. The gene is transcribed into polyadenylated mRNA which is most abundant in flowers, and more frequent in tubers than in leaves. The amino acid sequence is well conserved relative to the nuclear-encoded 75 kDa and 78 kDa subunits of Bos taurus and Neurospora crassa, respectively, and to the Paracoccus denitrificans homologue, most prominently in the region presumed to carry the iron-sulphur clusters. Polyclonal antibodies directed against the 78 kDa complex I subunit of N. crassa recognise the 76 kDa polypeptide in potato mitochondrial complex I, and additionally a polypeptide of 75 kDa in solubilised stroma thylakoids from spinach chloroplasts. The 32 amino acid residues long presequence of the potato mitochondrial 76 kDa complex I subunit targets the precursor polypeptide into isolated potato mitochondria but not into isolated chloroplasts. These results suggest that chloroplast stroma thylakoids contain a protein similar in size and antigenicity to, but genetically distinct from, the mitochondrial subunit.

A polypeptide subunit of complex I from Neurospora crassa, homologous to bovine TYKY, was expressed in Escherichia coli, purified and used for the production of rabbit antiserum. The mature mitochondrial protein displays a molecular mass of 21280 Da and results from cleavage of a presequence consisting of the first 34 N-terminal amino acids of the precursor. This protein was found closely associated with the peripheral arm of complex I.

Three monoclonal antibodies (mAbs), S4D1, S3B3 and S1E5, were produced from hybridoma cell lines raised from mice immunized with hyphal walls of Neurospora crassa and one (Pax-1) from mice immunized with hyphal walls of Paxillus involutus. In immunofluorescence studies, the three N. crassa mAbs recognized epitopes with different patterns of distribution at the hyphal surface of N. crassa. S4D1 recognized an epitope which was present on the surface of both conidia and hyphae; S3B3 recognized an epitope seen only at the ends of conidia or in the septal region of hyphae and conidial chains; and S1E5 recognized an epitope present on the surface of hyphae, but not on mature conidia. mAb Pax-1 reacted with hyphal wall fragments of Pax. involutus and with N. crassa conidia in a similar way to S3B3. S4D1 reacted with an epitope found in 1,3-alpha-glycan preparations from hyphal walls of different fungi. The surface distribution of this epitope varied: it was found on the surface of both conidia and hyphae of N. crassa and Aspergillus nidulans, on the basidiospore surface only of Amanita muscaria, and on the hyphae but not the conidia of Penicillium chrysogenum. Immunogold studies revealed that the epitope was present throughout the wall of conidia and hyphae of N. crassa. mAbs S3B3, S1E5 and Pax-1 also reacted with other fungi: for example Pax-1 cross-reacted with all fungi tested except for a member of the Zygomycota. Immunogold studies revealed that epitopes of these three mAbs were present within the inner layers of the walls of conidia and hyphae of N. crassa.

Polyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae CHS2 gene product were used to identify and localize chitin synthase in the filamentous ascomycete Neurospora crassa. A single band of approximately 110 kDa was observed in Western blots of total protein extracts of N. crassa, probed with these antibodies. However, several additional bands were labelled when membrane fraction proteins (microsomes) were probed. Histo-immunochemical localization of chitin synthase confirmed that the polypeptide is compartmentalized in membranous vesicles (chitosomes), which are abundant in the vicinity of the hyphal tip. TEM analysis did not reveal chitin synthase in the plasma membrane. However, dense labelling of membrane-associated chitin synthase was observed by light-microscopic analysis of N. crassa protoplasts and at young hyphal tips.

P59Nc is a polypeptide associated with bundles of cytoplasmic and nuclear filamentous structures of 8-10 nm of diameter in Neurospora crassa cells. It is immunologically unrelated to both higher and lower eucaryotic tubulin and actin proteins and is detected weakly by the anti IFA monoclonal antibody. We analyze here the immunological relationship between P59Nc and intermediate filament (IF) mammalian proteins by using anti P59Nc, anti keratin, anti vimentin and anti IFA antibodies. Anti P59Nc antibodies detected a 59 kDa polypeptide from rat and bovine tissues which copurifies with polypeptides of the IF family. Neither P59Nc nor the 59 kDa rat polypeptide were recognized by anti keratin or anti vimentin antibodies. Immunostaining of rat tongue sections with anti P59Nc antibodies showed that the 59 kDa rat polypeptide is present in the cortical cytoplasm of suprabasal epithelial cells. The results indicate that P59Nc shares common antigenic determinants with a still uncharacterized 59 kDa polypeptide from mammalian tissues which have extractability and immunological properties similar to those of IF polypeptides and shows a tissular distribution and a cellular localization similar but distinguishable from keratins.

The endonucleases from Neurospora crassa and Saccharomyces cerevisiae are not closely related antigenically. They also differ with respect to their activity at pH 8, their degree of hydrophobicity, and their sensitivity to elevated temperatures. However, the two nucleases have similar specific activities, are inhibited by EDTA, and have nearly identical substrate specificities. Since the enzymes also have the same mode of action and intracellular location, these similarities may indicate that they have the same physiological role despite their structural differences.

Antisera have been raised in rabbits against three wall fractions from Neurospora crassa. Fractions were separated according to Mahadevan & Tatum (1965), i.e. fraction I, glucan-peptide-galactosamine complex; fraction III, laminarin-like glucan; and fraction IV, chitin. Distinct patterns of immunofluorescent staining were obtained using an indirect staining method. Hyphae stained with antiserum to fraction I showed maximum fluorescence in the apical and/or subapical regions: in both cases, fluorescence showed a sharp decrease with distance behing the subapical region. Hyphae stained with antiserum to fraction III showed faintly fluorescent tips with fluorescence increasing with distance from the tip. Hyphae stained with antiserum to fraction IV showed faint fluorescence, equivalent to levels of autofluorescence, except at the sites of hyphal fractures. Antisera were also raised against whole walls from 24 and 120 h cultures. Hyphae stained with antisera against whole walls which had previously been absorbed to remove antibodies to fractions I, III, and IV showed preferential staining of apices. The uncharacterized tip antigen(s) thus revealed was also demonstrated on immunodiffusion plates. This pattern of immunofluorescence was compared to the fluorescence of apices after staining with an optical brightener. Enzymic dissection procedures did not generally give reliable results with apices from 24 h cultures. Untreated apices appeared amorphous, while a drastic chemical treatment revealed randomly oriented microfibrils which were shown to be alpha-chitin. The apical hyphal walls were significantly thinner than those from more mature hyphal regions.

Mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase, but, unlike nuc-1 and nuc-2 mutants, are able to make the repressible acid phosphatase and the repressible phosphate permease under conditions of derepression (phosphate deprivation). The new mutants, called pho-2, map in Linkage Group V, and are unlinked to the putative control mutants, nuc-1, nuc-2-pcon(c), and preg(c). Three of the pho-2 mutants do not make detectable amounts of repressible alkaline phosphatase, but the fourth makes about 1% of the level found in wild type. The small amount of alkaline phosphatase made by this strain appears to be qualitatively similar or identical to the wild-type enzyme, as judged by electrophoretic mobility, heat stability, and titration with specific antibody to the wild-type enzyme. Several revertants of this strain have been examined in the same way, and the alkaline phosphatase of these strains also appears to be qualitatively normal. Reversion events can occur at, or near, the pho-2 locus, but also occur in at least two unlinked sites (suppressor mutations). One suppressor maps very close to nuc-1.

Two major immunoreactive proteins of Mr 41,700 and 36,100 have been detected in crude mycelial extracts with polyclonal antibodies raised against arginase purified from Neurospora crassa. The latter corresponded to the protein used to obtain the antibodies. Both polypeptides were either missing or present in very low amounts in mutant strains having little or no detectable arginase activity. The relative proportion of the two species was altered in strains containing the nitrogen catabolite regulatory mutation nit-2. Peptide mapping indicated that the two species are very closely related, but several of the peptides which appeared to be identical by staining reacted differently with the antibodies. Both species were produced by in vitro translation of poly(A)+ mRNA, although the larger species was produced to a much smaller extent than was expected from its abundance in vivo. The results suggest the existence of multiple forms of arginase in N. crassa which differ in their response to nitrogen catabolite regulation.

Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species.

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987). Two mutants in the cyt-18 gene (cyt-18-1 and cyt-18-2) are defective in both mitochondrial protein synthesis and splicing, and an activity that splices the mitochondrial large rRNA intron copurifies with a component of mitochondrial tyrosyl-tRNA synthetase. Here, we used antibodies against different trpE-cyt-18 fusion proteins to identify the cyt-18 gene product as a basic protein having an apparent molecular mass of 67 kilodaltons (kDa). Both the cyt-18-1 and cyt-18-2 mutants contain relatively high amounts of inactive cyt-18 protein detected immunochemically. Biochemical experiments show that the 67-kDa cyt-18 protein copurifies with splicing and synthetase activity through a number of different column chromatographic procedures. Some fractions having splicing activity contain only one or two prominent polypeptide bands, and the cyt-18 protein is among the few, if not only, major bands in common between the different fractions that have splicing activity. Phosphocellulose columns resolve three different forms or complexes of the cyt-18 protein that have splicing or synthetase activity or both. Gel filtration experiments show that splicing activity has a relatively small molecular mass (peak at 150 kDa with activity trailing to lower molecular masses) and could correspond simply to dimers or monomers, or both, of the cyt-18 protein. Finally, antibodies against different segments of the cyt-18 protein inhibit splicing of the large rRNA intron in vitro. Our results indicate that both splicing and tyrosyl-tRNA synthetase activity are associated with the same 67-kDa protein encoded by the cyt-18 gene. This protein is a key constituent of splicing activity; it functions directly in splicing, and few, if any, additional components are required for splicing the large rRNA intron.

Trehalases from the ascospores of Neurospora tetrasperma and the mycelium of N. crassa were compared. Enzymes from both sources have identical electrophoretic mobilities, K(m)'s, responses to pH, immunological reactions, and activities in low-molarity buffers. Because both enzymes are so similar, conclusions about the properties of the ascospore enzyme may, be made by studying mycelial trehalase. Mycelial trehalase is most active and stable in low-molarity buffers. The enzyme exists in at least three species; the smallest has a molecular weight between 105,000 and 125,000 and is predominant in low-molarity buffers at 37 C. The stability of trehalase to heating at 65 C can be increased by increasing enzyme concentration and by the addition of polyols. Ascospores contain large amounts of trehalose, which protects trehalase from heat inactivation at 65 C. The importance of this phenomenon in vivo and its relationship to the localization of trehalase in ascospores is discussed.

An association of trehalase with the innermost wall (endosporium) of ascospores of Neurospora is suggested, because this enzyme could be lyophilized in the presence of various wall components and heated in this dried state at 65 C without loss of activity. Ground ascospore walls, purified mycelial walls, a wall fraction consisting of protein, glucan and polygalactosamine, or bovine serum albumin stabilize trehalase under these conditions. No other substances tested protected as well as the above materials. Immunofluorescent labeling of trehalase shows that it is localized in the endosporium. Therefore, it is most probable that in dormant ascospores of Neurospora, trehalase, and its substrate, trehalose, are physically separated. Trehalose is located in the cytoplasm, whereas trehalase resides within the protein and carbohydrate matrix of the innermost major cell wall layer of the ascospore. The association with the cell wall protects the enzyme against the heating which is necessary to activate germination. Activation, whether by heat or chemical treatment (furfural), probably involves an increase in the permeability of the ascospore plasma membrane allowing trehalose to diffuse to the vicinity of its hydrolase, thereby providing the energy and intermediates for germination.