Isolation and characterization of the tyrosinase gene from Neurospora crassa

A precursor form of Neurospora crassa tyrosinase has been identified by Western transfer from crude protein extracts and by immunoprecipitation of in vitro translated tyrosinase mRNA. The molecular weight of protyrosinase (75,000) exceeds that of mature tyrosinase (46,000) by about 50%. In order to deduce the primary structure and the nature of the extension, the tyrosinase gene was cloned. Poly(A) RNA isolated from tyrosinase-induced cultures of N. crassa was used as a template for cDNA synthesis, primed by a tyrosinase-specific, 32-fold degenerate heptadecanucleotide. Based on this sequence, a unique 21-mer was synthesized and used to screen a cDNA library constructed from tyrosinase-enriched mRNA. A partial genomic DNA library from wild-type strain TS and a genomic library from strain OR were screened using a 400-base pair nick-translated SalI fragment from a tyrosinase-positive cDNA clone as hybridization probe. The DNA sequences obtained revealed the presence of two allelic forms of this enzyme. The coding regions are interrupted by two short introns, of 52 and 99 base pairs. The encoded proteins differ in 3 out of 621 amino acid residues. A comparison of the deduced amino acid sequence with the known primary structure of mature tyrosinase alleles (Rüegg, C., Ammer, D., and Lerch, K. (1982) J. Biol. Chem. 257, 6420-6426) showed that the enzyme is synthesized as a precursor. Protyrosinase exceeds the mature protein by 213 amino acids at its carboxyl terminus. The possible involvement of carboxyl-terminal processing in enzyme activation is discussed.     

Role of sulfhydryl compounds in the control of tyrosinase activity in Neurospora crassa

It is known that Neurospora crassa mycelia cultured in standard concentrations (76 to 190 µg/ml) of sulfate accumulate a low molecular weight inhibitor of tyrosinase (monophenol, dihydroxyphenylalanine: oxygen oxidorenductase; EC 1.14.1.18.1.). This is not observed in cultures grown under sulfate-limiting conditions. The chemical nature of tyrosinase inhibition was investigated. It was shown to be due to the low molecular weight sulfhydryl fraction of the extracts, in which glutathione is predominant. The concentration of low molecular weight sulfhydryl compounds decreased sharply in mycelia submitted to various treatments which also derepressed tyrosinase, such as (i) starvation in phosphate buffer, (ii) treatment with cycloheximide, and (iii) mating. These results suggest that the concentration of sulfhydryl compounds may be of physiological significance in the control of tyrosinase activity in N. crassa.     

Purification and characterization of arginase from Neurospora crassa

We have purified an enzymatically active form of arginase from a wild-type strain of Neurospora crassa to homogeneity. The enzyme has a subunit molecular weight of 38,300 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein migrated as a hexamer during gel-filtration chromatography with an apparent molecular weight of 266,000. The enzyme exhibited hyperbolic kinetics at pH 9.5 with an apparent Km for arginine of 131 mM. Antiserum was prepared against the purified enzyme and used to demonstrate the existence of three cross-reactive proteins in crude extracts of wild-type N. crassa. One of these proteins corresponded to the purified protein, whereas the other two were of molecular weights 41,700 and 26,800, respectively. Using the same antiserum, we found that rat liver, but not rat kidney, contains immunoreactive material. We also detected two proteins in extracts of Saccharomyces cerevisiae that were weakly cross-reactive with the antiserum. These data provide evidence for the existence of multiple forms of arginase in fungi as well as in mammals.     

Purification and some properties of NAD-glycohydrolase from conidia of Neurospora crassa

NAD-glycohydrolase from conidia of Neurospora crassa was purified by affinity chromatography, using 4-methylnicotinamide adenine dinucleotide as ligand immobilized onto Sepharose through a hydrophilic spacer arm. The pure enzyme is a glycoprotein with an isoelectric point of 5.5 and a molecular weight of 33 000 as determined by sodium dodecylsulphate gel electrophoresis. The specific activity is the highest so far found for NAD-glycohydrolases and in various aspects the enzyme is different from that isolated from mycelia of N. crassa grown in a 'zinc-deficient' medium.     

The beta-oxidation system in catalase-free microbodies of the filamentous fungus Neurospora crassa. Purification of a multifunctional protein possessing 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase activities.

A trifunctional beta-oxidation protein, designated TFP, was purified to apparent homogeneity from oleate-induced mycelia of Neurospora crassa. 2-Enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase activities copurified in constant ratios with this protein when crude extracts were subjected to cation-exchange, dye-ligand, and adsorption chromatography. Trifunctionality was substantiated by coinciding enzyme activity ratios during the last two purification steps and additional chromatographic steps. The enzyme was shown to be a 365-kDa tetramer of subunits with a molecular mass of 93 kDa. Several lines of evidence suggest that these subunits are identical. Monospecific antibodies raised against the homogenous protein specifically precipitated the three enzymatic activities of TFP. Immunoblotting of fractions obtained after sucrose density gradient centrifugation of a crude extract indicated that TFP was exclusively localized in glyoxysome-like microbodies. The beta-oxidation system of N. crassa is structurally related to those of peroxisomes despite the presence of an acyl-CoA dehydrogenase rather than an acyl-CoA oxidase. A mitochondrial 2-enoyl-CoA hydratase activity was separated from TFP and purified to apparent homogeneity. The absence of all other beta-oxidation activities from mitochondria suggests that this organelle and its 2-enoyl-CoA hydratase are not involved in fatty acid degradation in N. crassa.     

Transient Expression of High Molecular Weight,Heat Sensitive,Trypsin-Resistant Form of Tyrosinase in B-16 Melanoma Cells

High molecular weight forms of tyrosinase have been found to be expressed during spontaneous remelanization of the amelanotic B-16 melanoma cells in culture as well as in melanotic tumors formed from amelanotic melanoma cells grown in C57BL/6J mice. Overnight extraction of the crude melanosomal fractions from such tumors and cultured melanoma cells reveal the presence of an additional DOPA-MBTH positive band well below the stacking gel. This band has been found to be α-PEP7 (antibody specific for tyrosinase) positive and α-PEPl (antibody specific for TRP-1) negative on Western blot analysis. Heat treatment at 60°C for 60 min results in the loss of this band and considerable loss of activity of the melanosomal extract. Trypsin treatment of these melanosomal extracts resulted in a minor change in the mobility of the high molecular weight band. SDS-PAGE under reduced conditions followed by Western blotting revealed that the high molecular weight band was lost and not detected by α-PEP7 or α-PEPl. These findings indicate that high molecular weight, heat sensitive and trypsin resistant forms of tyrosinase are transiently expressed in B-16 melanoma cells and tumors that are initiating remelanization following phenotypic drift towards the amelanotic state.     

Immunochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs.

Glutamine synthetase derived from two Neurospora crassa glutamine auxotrophs was characterized. Previous genetic studies indicated that the mutations responsible for the glutamine auxotrophy are allelic and map in chromosome V. When measured in crude extracts, both mutant strains had lower glutamine synthetase specific activity than that found in the wild-type strain. The enzyme from both auxotrophs and the wild-type strain was partially purified from cultures grown on glutamine as the sole nitrogen source, and immunochemical studies were performed in crude extracts and purified fractions. Quantitative rocket immunoelectrophoresis indicated that the activity per enzyme molecule is lower in the mutants than in the wild-type strain; immunoelectrophoresis and immunochemical titration of enzyme activity demonstrated structural differences between the enzymes from both auxotrophs. On the other hand, the monomer of glutamine synthetase of both mutants was found to be of a molecular weight similar to that of the wild-type strain. These data indicate that the mutations are located in the structural gene of N. crassa glutamine synthetase.     

THE pH STABILITY OF PROTYROSINASE AND TYROSINASE

1. pH stability diagrams for protyrosinase and for tyrosinase were constructed. 2. Above pH 7.30 protyrosinase is unstable. Between pH 7.30 and pH 9.30 there is a partial destruction. Beyond pH 9.30 it changes irreversibly into tyrosinase which in turn is destroyed beyond pH 10.12. 3. Through the lower ranges of pH protyrosinase is less stable than tyrosinase The former is destroyed below pH 4.80, while the latter is unaffected until the pH drops below 4.10. 4. The tyrosinase produced at high pH values resembles that produced by other methods.     

NH2-Terminal Residues of Neurospora crassa Proteins

The NH(2)-terminal amino acid composition of the soluble and ribosomal proteins from Neurospora crassa mycelia and conidia was determined by the dinitrophenyl method. A nonrandom distribution of NH(2)-terminal amino acids was observed in the complex protein mixtures. Glycine, alanine, and serine accounted for 75% of the NH(2)-terminal amino acids, and glycine appeared most frequently in mature proteins of mycelia. The appearance of phenylalanine as one of the major NH(2)-termini in crude conidial fraction suggests that the composition of proteins may vary in different developmental stages.     

Levels of sulfhydryls and disulfides in proteins from Neurospora crassa conidia and mycelia

Proteins extracted with 6 M guanidine at 90 degrees C from conidia (asexual spores) of Neurospora crassa contained ca. 25% more total protein thiol and a fivefold-higher content of disulfide bonds than proteins extracted from mycelia, as determined by labeling with iodo[14C]acetic acid. The total thiol content was 88 mumol/g of protein in conidia and 70 mumol/g of protein in mycelia. The level of protein disulfide was 18.5 mumol/g of protein in conidia and 3.5 mumol/g of protein in mycelia, by the iodo[14C]acetic acid labeling method. Confirmatory results were obtained with 5'5-dithio-bis-2-nitrobenzoic acid titration of protein thiol groups in 1% sodium dodecyl sulfate as well as by amino acid analysis of cysteic acid derivatives. Buffer-extracted proteins from conidia, but not mycelia, were found to contain enriched levels of protein thiols and disulfides per gram of protein as compared with guanidine hydrochloride extracts. It was demonstrated that the high disulfide content of crude conidial extracts was not due to measurable levels of mixed disulfides formed between protein sulfhydryl groups and cysteine. During germination of the conidia, the high disulfide levels of the conidial proteins remained constant. These data suggest that, unlike the disulfides of glutathione, the bulk of conidial protein disulfides were not reduced, excreted, or extensively degraded during germination.     

Acidic ribosomal proteins of Neurospora crassa

Neurospora crassa acidic ribosomal proteins from the high salt-ethanol extract of 80 S ribosomes have been fractionated by DEAE-cellulose chromatography. Six acidic ribosomal proteins were purified. All resemble Escherichia coli L7 and L12 in amino acid composition and molecular weight but each has a slightly different net charge at pH 3.2. Four have an apparent molecular weight of approx. 14 000, and two have a molecular weight of approx. 14 800. The amino acid compositions and circular dichroism (CD) spectra of the purified Neuropsora proteins are identical for the four 14 kDa proteins, but clearly distinguishable from the two 14.8 kDa proteins. The latter are also identical in amino acid composition and CD spectra. This suggests that there are two Neurospora acidic, or 'A', proteins, one of which exists in four microheterogeneous forms and the other exists in two forms.     

Characterization of two allelic forms of Neurospora crassa laccase. Amino- and carboxyl-terminal processing of a precursor

The complete structures of the laccase genes isolated from two different Neurospora crassa wild-type strains are described. The genes were cloned by screening partial genomic DNA libraries with a nick-translated laccase-specified 1.36-kilobase SalI fragment (Germann, U. A., and Lerch, K. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8854-8858) as a hybridization probe. Nucleotide sequence analysis revealed the presence of two different allelic forms. They conform to the same structural organization, but show an overall divergence of 5.3% which is mainly the result of point mutations in the nontranslated regions. The coding parts are interrupted by a short intron. The encoded proteins differ in 12 out of 619 amino acid residues. A comparison of the primary structure deduced from the nucleotide sequence of the gene with a protein chemical analysis of the two terminal cyanogen bromide fragments of extracellular N. crassa laccase revealed that the enzyme is synthesized as a precursor. The precursor protein exceeds the mature protein by 49 amino acids at its amino terminus and by 13 amino acids at its carboxyl terminus, thus indicating a complex maturation pathway. The possible involvement of amino-terminal processing in secretion and of carboxyl-terminal processing in activation of the enzyme is discussed.     

Sulfate transport in Neurospora crassa: regulation, turnover, and cellular localization of the CYS-14 protein.

Uptake of inorganic sulfate in Neurospora crassa is governed by the sulfur regulatory circuit and is under the control of positively and negatively acting regulatory genes. Two genetically and biochemically distinct systems are responsible for the uptake of sulfate from the environment. One of these, sulfate permease II, encoded by the cys-14 gene, functions primarily in mycelia. A defined region of the CYS-14 protein was highly expressed in Escherichia coli and purified. Anti-CYS-14 antibody was produced and used to detect the CYS-14 protein in N. crassa extracts. The CYS-14 protein has an approximate molecular weight of 95K, in agreement with its calculated size based on its predicted amino acid sequence. The steady-state level of the CYS-14 protein is highly regulated in wild-type mycelia and constitutive in an scon-1 mutant, whereas no CYS-14 protein could be detected in a cys-3 mutant. Following the accumulation of the cys-14 mRNA, that reaches its maximum in about 6 h, the CYS-14 protein accumulates to a maximum level in about 8 h after derepression. During conditions of sulfur repression, the CYS-14 protein turns over with a half-life of approximately 2 h. The CYS-14 protein appears to be localized in the plasma membrane, suggesting that it functions as a sulfate ion transporter.     

Soluble adenylate cyclase activity in Neurospora crassa.

A soluble form of adenylate cyclase was extracted from mycelia of Neurospora crassa wild-type strains. This enzyme activity was purified by chromatography on hexyl-amino-Sepharose, agarose and Blue Sepharose and preparative polyacrylamide-gel electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, peak fractions from the later purification steps showed a main polypeptide band with an apparent molecular weight of about 66 000. The following hydrodynamic and molecular parameters were established for the Neurospora soluble adenylate cyclase activity: sedimentation coefficient, 6.25 S; Stokes radius, 7.3 nm; partial specific volume, 0.74 ml/g; molecular weight, 202 000; frictional ratio, 1.65. The isoelectric point of this enzyme activity was 4.65. The enzyme was not activated by GTP, [beta gamma-imido]GTP, fluoride or cholera toxin.     

Immunopurification of highly specific antibodies to calmodulin from Neurospora crassa

Abstract Highly specific antibodies against calmodulin from Neurospora crassa were produced in rabbits. These antibodies were immunopurified by chromatography on Neurospora calmodulin-Sepharose, and had a titer for purified calmodulin from Neurospora crassa and bovine testis of 80 ng/ml and 2.5 μg/ml respectively. By immunoblot, as little as 8 ng of pure Neurospora calmodulin could be detected, and the antibodies revealed calmodulin in crude homogenates from Neurospora crassa and Aspergillus nidulans while in homogenates from Allomyces arbuscula, Saccharomyces cerevisiae, Dictyostelium discoideum and bovine testis, calmodulin remained undetected.     

The major intracellular alkaline deoxyribonuclease activities expressed in wild-type and Rec-like mutants of Neurospora crassa.

Over 95% of the deoxyribonuclease (DNase) activity of log-phase mycelia of Neurospora crassa is expressed as single-strand (ss) specific endonucleolytic activity. This activity is associated with three nucleases (D1, D2, and D3) which after partial purification from extracts, express activity with double-strand (ds) DNA as well. All three enzymes also degrade RNA at approximately the same rates that they degrade ss-DNA. D3 has been identified as endoexonuclease, an enzyme previously shown to have endonuclease activity with ss-DNA and RNA and exonuclease activity with ds-DNA, both of which are inhibited by ATP. D3 is inhibited by ATP, is relatively resistant to p-hydroxymercuribenzoate (PHMB), and sediments with an apparent molecular weight of 75 000. D2 has the properties of the previously described mitochondrial nuclease. It is a relatively unstable Mg2+-dependent endonuclease with no appreciable strand specificity for DNA. In addition, it is not inhibited by ATP and is strongly inhibited by PHMB and by the ethylenediamine tetraacetic acid (EDTA). It also sediments with an apparent molecular weight of 75,000. The properties of D1 are quite variable from one preparation to another. Freshly isolated D1 sediments with an apparent molecular weight of 180 000. It often shows some inhibition by ATP, but is relatively resistant to both PHMB and EDTA. However, on 'ageing,' the properties of D1 gradually convert to those of D2 with concomitant decrease in molecular weight, loss of inhibition by ATP, and increase in sensitivities to PHMB and EDTA. The results indicate that D1 is very likely a second form of the mitochondrial enzyme. Evidence was obtained for the presence of protein inhibitor(s) in crude extracts which may account for the masking of the ds-DNase activities of these enzymes in extracts. Two Rec-like mutants of Neurospora (uvs-3, and nuh-4) are deficient mainly inexpressed levels of D3, the endo-exonuclease. However, the levels of inactive endo-exonuclease precursor in these two mutants are higher than in the wild type. There may, therefore, be some defect in the conversion of precursor to active enzyme in these two mutants. Another mutant, which is not sensitive to mutagens relative to the wild (nuh-3), has depressed levels of both endo-exonuclease and the mitochondrial enzyme. Nuh-3 has some defect in the conversion of D1 to D2. Proteinases probably play some role in vivo in these enzyme conversions.     

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.     

The functional unit of the arom conjugate in Neurospora

The functional unit of arom polyenzyme conjugate of Neurospora crassa was determined by analysis of radiation inactivation of each of the five activities in the conjugate. The functional targets for all five enzymes were in close agreement with the value of 300,000 obtained by conventional hydrodynamic procedure for the native dimeric structure. These data indicate that at least 95% of the functional enzyme system in crude extracts exists in a dimeric form and that both polypeptide chains of the homodimer are required for full activity of each of the five enzymes.     

Occurrence of N-formylkynurenine in extracts of Neurospora crassa: evidence for the activity of tryptophan pyrrolase

N-formylkynurenine and kynurenine have been detected in extracts of tryptophan-grown Neurospora crassa. When the mycelia were grown in medium supplemented with l-[2-(14)C]tryptophan, the radioactivity was detected in N-formylkynurenine and N-formylanthranilic acid; with l-[beta-(14)C]tryptophan, radioactivity was detected in N-formylkynurenine, kynurenine, kynurenic acid, and xanthurenic acid. The occurrence of N-formylkynurenine in extracts of tryptophan-grown Neurospora is interpreted as direct evidence for the activity of tryptophan pyrrolase in this organism. The presence of this enzyme was expected on the basis of several earlier studies, but its activity in vitro has so far escaped detection. The in vivo evidence presented here suggests its presence and contributes importantly to our understanding of the tryptophan-anthranilic acid cycle.