Multiple intracellular peptidases in Neurospora crassa.

Neurospora crassa possesses multiple intracellular peptidases which display overlapping substrate specificities. They were readily detected by an in situ staining procedure for peptidases separated in polyacrylamide gels, within which the auxilliary enzyme, l-amino acid oxidase, was immobilized. Eleven different intracellular peptidases were identified by electrophoretic separation and verified by their individual patterns of substrate specificities. Most peptide substrates tested were hydrolyzed by several different peptidases. The multiple intracellular peptidases may play overlapping roles in several basic cell processes which involve peptidase activity. The amount of peptidase activity for leucylglycine present in crude extracts of cells grown under widely different conditions was relatively constant, suggesting that this enzyme may be constitutive, although alterations in the amounts of individual peptidase isozymes may occur. A single enzyme, designated peptidase II, was partially purified and obtained free from the other peptidase species. Peptidase II was found to be an aminopeptidase with activity toward many peptides of varied composition and size. It was more active with tripeptides than homologous dipeptides and showed strong activity toward methionine-containing peptides. This enzyme, with a molecular weight of about 37,000, was thermolabile at 65 degrees C and was strongly inhibited by p-hydroxymercuribenzoate, Zn(2+), Co(2+), and Mn(2+), but was insensitive to the serine protease inhibitor phenylmethylsulfonyl fluoride. Peptidase II apparently possesses an essential sulfhydryl group and may be a metalloenzyme.     

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

Evidence for distinct kynureninase and hydroxykynureninase activities in Neurospora crassa

Previous studies have indicated that a single enzyme, "kynureninase," catalyzes the reactions of l-kynurenine to anthranilate and l-3-hydroxykynurenine to 3-hydroxyanthranilate in Neurospora crassa and in other organisms. The present report describes separate enzymes which catalyze these reactions in N. crassa. The first, a kynureninase, preferentially catalyzes kynurenine to anthranilate and is induced over 400-fold by tryptophan or a catabolite of tryptophan. The second, a hydroxykynureninase, is constitutive or noninducible by tryptophan and preferentially catalyzes l-3-hydroxykynurenine to 3-hydroxyanthranilate. The physiological significance of these enzymes may be inferred from the facts that (i) the noninducible enzyme hydroxykynureninase appears to be the main enzyme present in uninduced cells that is capable of catalyzing l-3-hydroxykynurenine to 3-hydroxyanthranilate for the indispensible synthesis of nicotinamide adenine dinucleotide, and (ii) the inducible enzyme kynureninase is induced by tryptophan to a concentration far in excess of that needed to meet the requirements of the cells for nicotinamide adenine dinucleotide, resulting in the excretion of anthranilate into the medium.     

Evidence for No Proteolytic Processing during Transport of Isocitrate Lyase into Glyoxysomes in Castor Bean Endosperm

The amino-terminal sequence of isocitrate lyase purified fromcastor bean endosperm glyoxysomes was compared with that deducedfrom the nucleotide sequence of cDNA for the enzyme [Plant Mol.Biol. (1987) 8: 471]. The isolated active enzyme lacked sixamino acid residues in the amino terminus, although the enzymeimmunoselected from a tissue homogenate with trichloroaceticacid had the amino-terminal part. Thus the six amino acid residuesseem to be eliminated during enzyme purification and the enzymeis transported into glyoxysomes without proteolytic processing. (Received August 31, 1987; Accepted November 30, 1987)     

Multiple Alterations in Metabolite Uptake in a Mutant of Neurospora crassa

A temperature-conditional lethal mutant of Neurospora crassa, un-t (55701), was resistant to neutral amino acid analogues by virtue of a decreased ability to transport these analogues and their natural congeners across the cell membrane. The uptake of acidic, but not basic, amino acids was also impaired, as was the uptake of potassium ions. After preincubation above the tolerated temperature, the ability to take up a still wider variety of metabolites was greatly reduced. Protoplasts of the mutant were more osmotically fragile than those of wild type. The possibility that the mutant has a generalized membrane defect is discussed.     

Nuclear endo-exonuclease of Neurospora crassa. Evidence for a role in DNA repair

The major nuclease activity in nuclei of mycelia of Neurospora crassa has been identified as that of endoexonuclease, an enzyme purified and characterized previously from mitochondria and vacuoles which acts endonucleolytically on single-stranded DNA and RNA and possesses highly processive exonuclease activity with double-stranded DNA. Cross-contamination from the other organelles was eliminated as a source of the activity. Endo-exonuclease of nucleoplasm, chromatin, and nuclear matrix showed 80-100% cross-reaction with antisera raised to purified extranuclear endoexonuclease and was also strongly inhibited by 20 microM aurin tricarboxylic acid. In addition, it yielded some of the same-sized polypeptides on activity gel analysis. Nuclei also contained immunochemically cross-reactive trypsin-activable endo-exonuclease activity, a form of enzyme that was shown previously to occur in high amounts in the cytosol and in a tightly bound form associated with the mitochondrial inner membrane. Pretreatment of wild-type mycelia for 1 h with 4-16 micrograms/ml the DNA-damaging agent, 4-nitroquinoline-1-oxide (4-NQO), which caused about 50-80% growth inhibition, resulted in a dose-dependent loss of up to 80% of inactive endo-exonuclease from nuclei. At low doses of 4-NQO, this was accompanied by increases in the level of active enzyme. Nuclei of the DNA repair-deficient uvs-3 mutant were found to contain only 12% of the active enzyme and about 32% of inactive enzyme as that in wild-type nuclei. Mycelial growth of this mutant was 10 times more sensitive to 4-NQO than the wild-type. At a dose which resulted in equivalent growth inhibition, 4-NQO had no effect on the level of active endo-exonuclease in uvs-3 nuclei and caused an increase (over 30%) in the level of inactive enzyme. These data are consistent with a role of endo-exonuclease in the repair of nuclear DNA.     

Acetylglutamate kinase-acetylglutamyl-phosphate reductase complex of Neurospora crassa. Evidence for two polypeptides

Mutations at the arg-6 locus in Neurospora crassa are divided into two complementation groups (A and B) and a third noncomplementing group. There are many suppressible nonsense mutations among mutants in complementation group B and one in the noncomplementing group; no nonsense mutations exist among mutants in complementation group A (Davis, R. H., and Weiss, R. L. (1983) Mol. Gen. Genet. 192, 46-50). We show here that the mutants are defective in either or both of two enzymes of arginine biosynthesis, acetylglutamate kinase and/or acetylglutamyl-phosphate reductase. Mutants in complementation group A lack acetylglutamate kinase, those in complementation group B lack acetylglutamyl-phosphate reductase, and those in the noncomplementing group lack both activities. Mutants in group B also have reduced levels of acetylglutamate kinase. The enzymes from purified mitochondria are readily separable by gel filtration and by Blue A dye affinity chromatography. Acetylglutamate kinase appears to be an octamer with a molecular weight of 400,000, whereas acetylglutamyl-phosphate reductase appears to be a dimer with a molecular weight of 93,000. This suggests that the two activities reside on distinct polypeptides. These results are best accommodated by the following model: the arg-6 locus encodes a single mRNA which is translated into a single polypeptide; the latter is then cleaved post-translationally to yield two physically separable enzymes.     

The Kinetics of Isocitrate Lyase Formation in Chlorella: Evidence for the Promotion of Enzyme Synthesis by Photophosphorylation

The addition of acetate to aerobic Chlorella pyrenoidosa indarkness was followed by the formations of isocitrate lyaseactiity. After a lag period of 40 minutes the formation proceededat a constant rate. By use of actylamide gel electrophoresisit was shown that the increase in enzyme activity was accompaniedby the formation of a new protein which, after separation byelectrophoresis, contained isocitrate lyase activity. The formationof isocitrate lyase was repressed by glucose; it was repressedby light in the presence of carbon dioxide, but not when DCMUwas added. In light, plus DCMU, isocitrate lyase was formedanaerobically and the capacity for photo-formation of isocitratelyase was saturated at 500 ergs/cm²/sec. In this respect theprocess resembled the photo-conversion of glucose to polysaccharidebut differed from the photo-assimilation of carbon dioxide whichbecame saturated at a heigher light intensity. Monochromaticlight of 706 mµ wavelength supported both isocitrate layseformation and the conversion of glucose to polysaccharide butnot carbon dioxide fixation. It is concluded that ATP generatedby cyclic photophosphorylatin can provide the energy for isocitratelyase synthesis in Chlorella.     

Conidiation in Neurospora crassa

Summary Conidiation in Neurospora crassa has been studied in vivo by time-lapse microphotography and shown to be most generally (in aerial, dry conditions) a budding-fission process. Such a two-phase process is characterized by an initial basifugal budding of proconidial elements which are then secondarily separated as maturing conidia by interconidial septa. Dry macroconidia of Neurospora are thus blasto-arthrospores, i.e. blastospores basifugally budded on conidiophores and secondarily disarticulated from the proconidial chain as arthrosporal elements. Inception and median splitting of the interconidial septum have been electron microphotographed.In the vegetative hyphae, ethanol dehydrogenase has been cytochemically detected by oxidative assay and demonstrates a dense, uniform distribution of activity except at the hyphal tips. In the conidiating hyphae, the ethanol dehydro-genase becomes less dense in distribution, especially in the budding apices. Cytochrome oxidase activity, localized in the mitochondria, is confined in the subapical zone of vegetative hyphae while at the initiation of conidiation it becomes dispersed throughout the proconidial buds.     

Photoperiodism in Neurospora crassa

Plants and animals use day or night length for seasonal control of reproduction and other biological functions. Overwhelming evidence suggests that this photoperiodic mechanism relies on a functional circadian system. Recent progress has defined how flowering time in plants is regulated by photoperiodic control of output pathways, but the underlying mechanisms of photoperiodism remain to be described. The authors investigate photoperiodism in a genetic model system for circadian rhythms research, Neurospora crassa. They find that both propagation and reproduction respond systematically to photoperiod. Furthermore, a nonreproductive light-regulated function is also enhanced under certain photoperiodic conditions. All of these photoperiodic responses require a functional circadian clock, in that they are absent in a clock mutant. Night break experiments show that measuring night length is one of the mechanisms used for photoperiod assessment. This represents the first formal report of photoperiodism in the fungi.     

Occurrence of Isocitrate Lyase in a Thermophilic Bacillus Species

A thermophilic, sporeforming bacterium has been isolated from soil on a medium containing acetate as a carbon source. This organism is similar to Bacillus stearothermophilus in most respects but differs in its inability to hydrolyze starch. Isocitrate lyase is present in cell-free extracts of organisms grown in a medium with acetate as a carbon source. The specific activity was 400 times lower in extracts of organisms utilizing glucose as a carbon source. With crude extracts, enzyme activity was strongly stimulated by Mg(++), but cysteine and ethylenediaminetetraacetate had little effect. It appeared to be more heat-stable than the pure isocitrate lyase from Pseudomonas indigofera.     

Effect of 6-Phosphogluconate on Isocitrate Lyase Activity

Abstract

Some characteristics of the inhibition of yeast isocitrate lyase by 6-phosphogluconate have been investigated. The existence of such an inhibition suggests that the level of 6-phosphogluconate may have a role in the metabolic control in the yeast cell of glucogenesis from acetyl-CoA, via the glyoxylate cycle.     

Isocitrate lyase from Neurospora crassa: pH dependence of catalysis and interaction with substrates and inhibitors.

The maximal velocity, V, for isocitrate cleavage by isocitrate lyase from Neurospora crassa is dependent on two dissociable groups with pK_a values of 6.1 and 8.6. A dissociable group with a pK_a of 8.5 on the enzyme-substrate complex affects the pK_m for isocitrate. The pK_i for homoisocitrate is affected in a like manner. The pH dependence of the pK_i's for succinate, a product of isocitrate cleavage, and the succinate analog maleate is similar to the pH dependence of the pK_m of isocitrate below pH 7.3, but is markedly different above this pH. Both the K_m for isocitrate and the K_i for succinate were dependent upon Mg²⁺ concentration. The pK_i for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, is dependent on a group with a pK_a of 6.8 on the enzyme-inhibitor complex. The pH dependence of the pK_i for phosphoenolpyruvate, which binds to the succinate site, suggests that it is dependent on two dissociable groups, one on phosphoenolpyruvate and one, by analogy to the pK_m for isocitrate, on the enzyme-glyoxylate-inhibitor complex.     

Induction of Isocitrate Lyase in Synchronous Cultures of Chlorella fusca

Isocitrate lyase (ICL) of Chlorella was induced with acetate, and induction kinetics followed in autospores and 6 h old cells of a synchronous culture. The enzyme could not be induced in illuminated cells. With both cell types 1.2 mM acetate was the optimal inducer concentration. Freeze-thawed cells and acetone powders were used for measurement of activity. With the former the time course of increase in activity was different at the two cell ages. With 6 h old cells the activity fluctuated: There was first a period of increase, then one with decrease and again one of increase. No such variation was found with freee-thawed autospores or with acetone powders of both cell stages. Darkening 6 h old cells for different periods of time before induction reduced the peak of activity, leaving the rate of the third phase unchanged. Illumination of darkened cells before induction increased the peak. Increasing the duration of both treatments increased their respective effects. Acetone extracts taken at different times after start of induction inhibited the ICL activity of a test preparation. The inhibition decreased concurrently with the variation in the ICL activity-found-when freee-thawed cells were used in the enzyme assay. The inducibility, taken as the rate of the third phase, was measured at different times during the 24 h synchronous cycle. Using three different acetate concentrations and both methods of cell preparation, we found that the inducibility was constant for 17 h whereafter it increased rapidly to a final level.     

Microconidia of Neurospora crassa

Neurospora crassa produces two types of vegetative spores-relatively small numbers of uninucleate microconidia and very large numbers of multinucleate macroconidia (blastoconidia and arthroconidia). The microconidia can function either as spermatia (male gametes) or as asexual reproductive structures or both. In nature they probably function exclusively in fertilization of protoperithecia. The environmental conditions favoring their formation and the pattern of their development are quite distinct from those of macroconidia. Mutants of N. crassa have been isolated in which macroconidiation is selectively blocked without affecting microconidiation, showing that these two types of conidial differentiation involve distinct developmental pathways. Unlike microconidia of some related ascomycetes, those of Neurospora are capable of germination, providing viable uninucleate haploid cells which are desired in several types of investigations. A technique of selectively removing macroconidia from culture initiated on cellophane overlying agar medium allows pure microconidia to be obtained even from the wild-type strains of Neurospora. The conditional microcyclic strain, mcm, allows either macroconidia or microconidia to be obtained at will, depending on the conditions of culture. The new methods of obtaining pure microconidia from normal laboratory strains will make it quick and easy to purify heterokaryotic transformants following introduction of DNA into multinucleate protoplasts. Moreover, these methods allow the detection of genetic variability that remains hidden within an individual fungus and the estimation of the frequency of nuclear types in laboratory-constructed heterokaryons. The discovery, function, and development of microconidia are described and their research applications are discussed in this review.