ATP-induced protyrosinase synthesis and carboxyl-terminal processing in Neurospora crassa

The effects of 3'-5' cyclic AMP and ATP upon tyrosinase induction in Neurospora crassa were examined. Northern analysis of total cellular RNA revealed rapid de novo synthesis of protyrosinase after addition of these substances to stationary-phase mycelia. The maturation of protyrosinase in crude extracts of mycelia was followed by Western analysis. Polyclonal rabbit antiserum directed against the denatured carboxyl-terminal extension of protyrosinase does recognize the proform and several intermediate forms of different molecular weight but not mature tyrosinase. Disruption of ATP-induced mycelia in sodium phosphate buffer (pH 6.0) demonstrate processing at the carboxyl-terminal end of protyrosinase. The activity assays revealed that protyrosinase is an inactive precursor and that at least two active forms of slightly different molecular weight are present in crude extracts. Maturation of protyrosinase thus involves specific and sequential proteolytic cleavage at the carboxyl-terminus. These results suggest the presence of a tyrosinase activator in Neurospora crassa mycelia, which is kept apart from protyrosinase in the intact mycelium.     

Purification and properties of Neurospora crassa laccase

Extracellular Neurospora laccase (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) has been purified to apparent homogeneity by classical purification techniques. The enzyme, which consists of mainly one form, has a molecular weight of 64,800 and contains 11% carbohydrate. The ultraviolet, visible, and electron paramagnetic resonance spectra indicate that both type I and type II copper are present, as described for the Polyporus versicolor enzyme. With the exception of phloroglucinol, only para- and ortho-diphenols serve as effective substrates for the enzyme. Like the extracellular form, intracellular laccase is a glycoprotein as shown by its ability to bind to Concanavalin A Sepharose. Other studies, including gel filtration and ion-exchange chromatography, revealed no differences between the intracellular and extracellular enzymes, suggesting that intracellular laccase is destined for excretion by the cell.     

Neurospora crassa mitochondria contain two forms of a 4'-phosphopantetheine-modified protein

When Neurospora crassa was labeled with [14C]pantothenic acid during growth, the mitochondrial fraction contained two bands of radioactivity of Mr 19,000 and 22,000 by sodium dodecyl sulfate gel electrophoresis. The 19-kDa band was converted to the 22-kDa band by four treatments which are characteristic of the cleavage of a thioester bond: dithiothreitol and 2-mercaptoethanol at basic but not neutral pH, alkaline methanolysis, sodium borohydride in tetrahydrofuran, and hydroxylamine at neutral pH. Mitochondrial subfractionation indicated that the 22-kDa form was preferentially associated with the soluble fraction while the 19-kDa form was found in all fractions. Several properties of the mitochondrial protein were similar to the Escherichia coli acyl carrier protein: Mr on sodium dodecyl sulfate gels, decreased electrophoretic mobility under deacylating conditions, isoelectric point, and covalent attachment of 4'-phosphopantetheine. The 19- and 22-kDa bands may therefore represent acylated and deacylated forms of a mitochondrial acyl carrier protein.     

Relationship between two major immunoreactive forms of arginase in Neurospora crassa.

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.     

Two forms of a mitochondrial endonuclease in Neurospora crassa.

Mitochondrial nuclease activity in Neurospora crassa occurs in membrane-bound and soluble forms in approximately equal proportions. These activities apparently are due to the same enzyme, which has an approximate molecular weight of 120 000. A portion of the insoluble enzyme appears to be associated with the inner mitochondrial membrane and is resistant to solubilization by detergent treatment as well as by physical disruption methods.     

Characterization of two abundantly expressed constitutive genes of Neurospora crassa

Two abundantly expressed, constitutive genes of Neurospora crassa were isolated during differential screening of Neurospora genomic libraries. The coding regions of these two genes, designated RLF1 and RLF3, were identified by hybridization of the cloned DNA sequences with cDNA probes made from polyadenylated RNA. The RLF3 gene was carried on a 15-kilobase Neurospora BamHI DNA fragment present in a lambda 1059 recombinant; a 2-kilobase restriction fragment that contains RLF3 was subcloned into plasmid pBR322 prior to further characterization. Southern blot analysis revealed that both RLF1 and RLF3 are single copy genes. Northern blot analysis and S1 nuclease mapping demonstrated that RLF1 is transcribed to yield a 1.6-kilobase RNA, whereas RLF3 appears to give rise to two distinct sized RNA species of 1.0 and 1.6 kilobases. RNA dot blot analysis provided conclusive evidence that both of these genes are constitutively expressed. These constitutive genes will be valuable to provide a detailed comparison with the 5' flanking regions of regulated genes.     

A single precursor protein for two separable mitochondrial enzymes in Neurospora crassa

The arg-6 locus of Neurospora crassa encodes two early enzymes of the arginine biosynthetic pathway, acetylglutamate kinase and acetylglutamyl-phosphate reductase. Previous genetic and biochemical analyses of this locus and its products showed that: 1) strains carrying polar nonsense mutations in the acetylglutamate kinase gene lacked both enzyme activities (Davis, R.H., and Weiss, R.L. (1983) Mol. Gen. Genet. 192, 46-50), and 2) the proteins isolated from mitochondria were completely separable (Wandinger-Ness, A., Wolf, E.C., Weiss, R.L., and Davis, R.H. (1985) J. Biol. Chem. 260,5974-5978). These data suggested that the two enzymes were initially synthesized as a single precursor which was subsequently cleaved into two distinct polypeptides. We report here the identification of a high molecular weight protein, synthesized in vitro from isolated N. crassa RNA, that contains sequences corresponding to acetylglutamate kinase as well as acetylglutamyl-phosphate reductase. An analogous precursor was identified in vivo by pulse-labeling experiments. The precursor was similar to other mitochondrial precursors in that its uptake and processing in vivo was rapid and required an intact mitochondrial electrochemical gradient. This represents the first report of a bifunctional protein precursor which gives rise to two mitochondrial enzymes.     

Intracellular localization of Neurospora crassa endo-exonuclease and its putative precursor.

Endo-exonuclease of rapidly growing mycelia of Neurospora crassa was found to be distributed in a ratio of about 1.6:1 in vacuoles and in mitochondria where it is associated with the inner membrane. Although the activity in vacuoles was readily released by osmotic shock, very little of that in mitochondria was released by this method. The mitochondrial activity was partially (60 to 70%) released by sonication, and the remaining activity was solubilized in the presence of Triton X-100. An inactive form of endo-exonuclease, activated in vitro by treatment with trypsin, is present in mycelia at a level over four times that of active enzyme. It was found to be distributed in a ratio of about 2.5:1 in the cytosol and in the inner membrane of mitochondria. The mitochondrial protein was more tightly bound than the active enzyme. Very little of the inactive enzyme was released by sonication, but it was solubilized in the presence of Triton X-100. The intracellular distribution of active and inactive forms of endo-exonuclease differs in a mutagen-sensitive mutant of Neurospora crassa (uvs-3) which shows many pleiotropic effects. The most striking difference in distribution is in the mitochondria where endo-exonuclease is present almost entirely in the inactive form at a level 30% higher than in wild-type mitochondria.     

Optimization of simultaneous production of tyrosinase and laccase by Neurospora crassa

Increasing demand for efficient and environmentally benign oxidation technologies has resulted in a focus on the use of oxidoreductases. Laccases and tyrosinases, which utilize molecular oxygen and produce water as by-product, are particularly attractive. Simultaneous production of laccase and tyrosinase was studied in Neurospora crassa FGSC #321 as the fungal strain which has the ability to produce tyrosinase intracellularly while producing laccase extracellularly. Using one-variable-at-a-time experiments and a Taguchi orthogonal L9 array demonstrated that a Vogel minimal medium containing 2.5% sucrose at pH 6.5 and 25?°C with no agitation or oxygen purging were the optimum conditions for N. crassa FGSC #321 growth. Conditions were adjusted to obtain the highest laccase and tyrosinase production. Results indicate that the control mechanisms for the production of both enzymes in N. crassa FGSC #321 are similar but not necessarily identical. Results revealed that transferring the harvested cells from the growth medium into the phosphate buffer (pH 6.8, 0.1M) containing cycloheximide (2?μM) and fluorouracil (2?mM) and increasing the temperature to 30?°C were the best conditions for simultaneous production of laccase and tyrosinase (1278 and 410?U/g of biomass, respectively). Nonetheless, starvation at 35?°C is proposed as the most cost-effective means for inducing laccase. The N. crassa laccase was characterized by using its molecular weight, pI value, optimal pH and temperature and stability.     

Characterization of Ribosomes from Neurospora crassa

Ribosomes isolated from growing hyphae of Neurospora crassa contain 53 per cent protein and 47 per cent RNA and have a sedimentation coefficient of 81S at 20°C and infinite dilution. These ribosomes are stable at pH 7.4 in the presence of 0.01 M and 0.002 M MgCl₂ but undergo a dissociation into smaller particles if the MgCl₂ concentration is lowered to 0.0001 M. Two types of RNA with sedimentation coefficients of 19S₂₀⁵⁰ and 13S₂₀⁵⁰ have been extracted from the 81S particles.     

Characterization of Neurospora crassa catabolic dehydroquinase purified from N. crassa and Escherichia coli.

1. Neurospora crassa catabolic dehydroquinase has been purified from N. crassa and Escherichia coli. 2. Protein-sequence and gel-electrophoretic data show that apparently pure, homogeneous native dehydroquinase is a mixture of intact and proteinase-cleaved enzyme monomers. 3. Protein-sequence data and steady-state kinetics show that the catabolic dehydroquinase gene of N. crassa is expressed with fidelity in E. coli.     

Characterization of a Novel Prevacuolar Compartment in Neurospora crassa

Using confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungus Neurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases in N. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts.