The type of mutations induced by carbon-ion-beam irradiation of the filamentous fungus Neurospora crassa

Heavy-ion beams are known to cause great damage to cellular components and are particularly renowned for their ability to generate DNA double-strand breaks (DSBs). To gain insight into the mutagenic effect of carbon-ion beams and how such damage is repaired by the cell, Neurospora crassa mutants deficient in one of three components involved in the repair of DSBs, nonhomologous end-joining (NHEJ), homologous recombination repair (HR), and the Mre11-Rad50-Xrs2 (MRX) complex, were irradiated with a carbon-ion beam and killing effect, mutation frequencies, and the type of mutation incurred by survivors were analysed. The sensitivity of the NHEJ-deficient strain (mus-52) was higher than that of the wild-type and the HR-deficient (mei-3) strains at low doses of radiation, but was little changed as the level increased. As a result both the wild-type and HR-deficient strains were more sensitive than the NHEJ-deficient strain at high radiation levels. In addition, the frequency of forward mutation at the adenine-3 (ad-3) loci of the NHEJ-deficient mutant was lower than that of the wild-type strain at all levels, while the mutation frequency of the HR-deficient strain was consistently ∼3-fold higher than the wild-type. From the comparison of mutation type of each strain, deletions were frequently observed in wild-type strain, whilst base substitution and deletion in the mus-52 and mei-3 strains. These mutations resulting from carbon-ion-beam irradiation depend on the mechanism invoked to cope with DSBs. Furthermore, in wild-type cells, these mechanisms likely compete to repair DSBs.     

The subcellular distribution of carotenoids in Neurospora crassa

The intracellular localization of carotenoids in the fungus Neurospora crassa was examined after completion of photoinduced biosynthesis of these pigments. Differential centrifugation of cell homogenates yielded subcellular fractions which were characterized by activities of several marker enzymes for cell constituents and in part purified by subsequent sucrose density gradient centrifugation. Most (ca 58%) of the carotenoids were found to be localized in lipid globules, but substantial amounts are also associated with two membrane fractions that were rich in membranes of the endoplasmic reticulum as indicated by high activities of NADPH- and NADH—cytochrome c reductase. These results, along with the coincidence in the distribution of both carotenoids and activities of specific marker enzymes in the sucrose density gradients, led to the conclusion that apart from lipid globules, carotenoids are also localized in membranes of the endoplasmic reticulum.     

Free and bound lipids of Neurospora crassa

Extraction of light and dark grown cells of Neurospora crassa with chloroform-methanol gave a free lipid extract in which the relative amounts and compositions of sterols, fatty acid and carotenoid fractions were determined. Further extraction of the cells with methanolic potassium hydroxide solution liberated a bound lipid fraction from the cells. The levels of the bound lipid fraction were much lower than those of the free lipids but analysis showed that the composition was similar to that of the free lipids.     

Subcellular site of carotenoid biosynthesis in Neurospora crassa

A cell-free system prepared from Neurospora crassa mycelia was capable of incorporating radioactivity from [¹⁴C]-mevalonic acid into phytoene and to a much lesser extent into more unsaturated carotenoids. Whereas carotenogenic activities were only minimal in extracts from dark-grown cultures, they were several-fold increased following a short in vivo illumination; this photo-induced increase was inhibited by cycloheximide. Subcellular localization of carotenogenic enzyme activities was investigated using incubations of particular isolated fractions, together with [¹⁴C]-mevalonic acid and a geranylgeranyl-pyrophosphate-synthesizing system provided by the endosperm of maturing pumpkin seeds. Maximum carotenogenic activity (ca 80%) was localized in two membrane fractions previously shown to contain plasma membranes and, in particular, membranes of the endoplasmic reticulum. The lipid layer, containing the bulk of carotenoid pigments, possesses only trace amounts of enzyme activities.     

C35-apocarotenoids in the yellow mutant Neurospora crassa YLO

The Neurospora crassa mutant YLO exhibits a yellow phenotype instead of the red-orange pigmentation of the wild type. Recently, it was shown that the mutant YLO is defective in a specific aldehyde dehydrogenase which catalyses the last step of carotenogenesis to the formation of neurosporaxanthin [Estrada, A.F., Youssar, L., Scherzinger, D., Al-Babili, S., Avalos, J., 2008. The ylo-1 gene encodes an aldehyde dehydrogenase responsible for the last reaction in the Neurospora carotenoid pathway. Mol. Microbiol. 69, 1207-1220]. Since different carotenoid compositions between wild type and YLO have been reported in earlier publications, the carotenoids of YLO were analyzed and unknown carotenoids identified. Fractionation of carotenoid extracts from YLO revealed in the less polar fraction two major carotenoids of low polarity which were found only in trace amounts in the wild type. Both carotenoids could be hydrolyzed with KOH to more polar products indicating the presence of fatty acid esters. The fatty acid moiety was identified as myristic acid by gas chromatography. Optical and mass spectra as well as co-chromatography with a synthesized authentic standard identified the free alcohols as 4′-apolycopene-4′-ol and 4′-apo-γ-carotene-4′-ol which assigns the dominating carotenoids in the YLO mutant as 4′-apolycopene-4′-myristate and 4′-apo-γ-carotene-4′-myristate. We can attribute the accumulation of these two carotenoids in YLO to the substantial mutation of the neurosporaxanthin-forming aldehyde dehydrogenase. However, the aldehyde intermediates 4′-apo-γ-carotene-4′-al and 4′-apo-lycopene-4′-al do not accumulate substantially but are reduced instead to the corresponding alcohols, 4′-apolycopene-4′-ol and 4′-apo-γ-carotene-4′-ol, and both further esterified with mainly myristic acid yielding 4′-apolycopene-4′-myristate and 4′-apo-γ-carotene-4′-myristate.     

Temperature-induced modifications of glycosphingolipids in plasma membranes of Neurospora crassa

Plasma membranes isolated from a cell-wall-less mutant of Neurospora crassa grown at 37 and 15°C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37°C membranes, while 15°C plasma membranes exhibited a ratio of nearly 2.0. Membranes formed under both growth conditions were found to contain glycosphingolipids. Cultures grown at the low temperature, however, were found to contain 6-fold higher levels of glycosphingolipids and a corresponding 2-fold reduction of phospholipid levels. The high glycosphingolipid content at 15°C compensates for the reduced levels of phospholipids in such a way that sterol/polar lipid ratios are almost the same in plasma membranes under the two growth conditions. Temperature-dependent changes in plasma-membrane phospholipid and glycosphingolipid species were also observed. Phosphatidylethanolamine levels were sharply reduced at 15°C, in addition to a moderate increase in levels of unsaturated phospholipid fatty acids. Glycosphingolipids contained high levels of long-chain hydroxy fatty acids, which constituted 75% of the total fraction at 37°C, but only 50% at 15°C. Compositional changes were also observed in the long-chain base component of glycosphingolipids with respect to growth temperature. Fluorescence polarization studies indicate that the observed lipid modifications in 15°C plasma membranes act to modulate bulk fluidity of the plasma-membrane lipids with respect to growth temperature. These studies suggest that coordinate modulation of glycosphingolipid, phospholipid and sterol content may be involved in regulation of plasma-membrane fluid properties during temperature acclimation.     

Characteristics of a transport-deficient mutant (nap) of Neurospora crassa

nap, previously characterized by Jacobson and Metzenberg as a neutral and acidic amino acid transport mutant, was found in this study to be reduced in transport activity for a variety of metabolites. Analysis of glycoprotein molecules involved in amino acid binding indicates that the deficiency is not at this level. The deficiency appears to be in some common component of all active transport systems.     

Effect of divalent metal ions on the synthesis of oligosaccharide side chains of Neurospora crassa glycoproteins

Neurospora crassa membrane preparations incorporated mannose from GDP-mannose-[¹⁴C] in the presence of Mg²⁺ into a polyprenol lipid and side chains of protein acceptor(s), which are labile on hydrolysis in weak base. The addition of Mn²⁺ to the reaction mixtures does not affect mannosyl lipid synthesis but it stimulates the transfer of mannose to larger oligosaccharide chains resistant to β-elimination and the transfer of a second mannosyl unit to form an O-glycosidically linked mannobiosyl side chain. Incubation of particulate preparations with polyprenol-mannose-[¹⁴C] in the presence of Mg²⁺ and Mn²⁺ also results in the transfer of a single mannose to the protein. When non-radioactive GDP-mannose is added to this reaction mixture, however, β-elimination yields mannobiose. The mannobiose is labeled in the reducing sugar only. These results indicate that the first mannose of this mannobiosyl side chain is transferred via a lipid intermediate, but the second mannose is transferred directly from GDP-mannose. In the presence of Mg²⁺ and Mn²⁺, mannose apparently is also transferred from polyprenol-mannose-[¹⁴C] to side chains which are resistant to hydrolysis.     

Mutational variants of the Neurospora crassa NADP-specific glutamate dehydrogenase altered in a conformational equilibrium

Seven defective variants of the NADP-specific glutamate dehydrogenase of Neurospora crassa, resulting from missense mutations in the am gene, are quantitatively different from the wild type enzyme in the allosteric equilibrium between enzymically active A and inactive I conformations, and in the kinetics of conformational transitions between these states. These abnormalities have been defined using measurements of enzymic activity and of the intrinsic tryptophan fluorescence emission of the proteins.The protein from am¹(Ser336 → Phe) is hyperstable in the A conformation but this state is enzymically inactive because it fails to bind coenzyme. The other six variants are potentially active but are, to different extents, hyperstable in the I conformation. They form a series of analogues, those of am¹³¹ (substitution not determined), am¹³⁰(Pro75 → Ser), am³(Glu393 → Gly), am²(His142 → Gln), am¹⁹(Lys141 → Met) in order of increasing abnormality of the equilibrium position. am¹²²(Trp389 changed to an undetermined residue) resembles am¹⁹. The hyperstability is sufficient to explain the auxotrophy of am The proteins of am¹³¹ and am¹³⁰ are, in addition, abnormally prone to denaturation. These hyperstabilities of the I state are small in free energy terms, consistent with the fact that the defects of some variants may be corrected or partially corrected by second site substitutions or by complementation in hybrid hexamers with am¹ protein.Five out of seven amino acid substitutions known to affect this equilibrium (including Gln391 → Arg of revertant am¹⁹24) involve charged residues clustered around positions 141 and 391. Interactions between these two parts of the polypeptide are implicated in stabilizing the A state of the enzyme, possibly by providing protonatable groups or part of the dicarboxylate binding site, and in affecting the environment of a tryptophan residue responsible for the fluorescence difference of the two conformations.     

Effects of inhibitors on the plasma membrane and mitochondrial adenosine triphosphatases of Neurospora crassa

A comparative study has been made of the effects of a variety of inhibitors on the plasma membrane ATPase and mitochondrial ATPase of Neurospora crassa. The most specific inhibitors proved to be vanadate and diethylstilbestrol for the plasma membrane ATPase and azide, oligomycin, venturicidin, and leucinostatin for mitochondrial ATPase. $\text{N,N′-}\text{Dicyclohexylcarbodiimide}$, octylguanidine, triphenylsulfonium chloride, and quercetin and related bioflavonoids inhibited both enzymes, although with different concentration dependences. Other compounds that were tested (phaseolin, fusicoccin, deoxycorticosterone, alachlor, salicyclic acid, $\text{N-1-}\text{napthylphthalamate}$, triiodobenzoic acid, cyclic AMP, cyclic GMP, theobromine, theophylline, and histamine) had no significant effect on either enzyme. Overall, the results indicate that the plasma membrane and mitochondrial ATPases are distinct enzymes, in spite of the fact that they may play related roles in H⁺ transport across their respective membranes.     

Characterization of the Neurospora crassa DHN melanin biosynthetic pathway in developing ascospores and peridium cells

Neurospora crassa contains all four enzymes for the synthesis of DHN (dihydroxynaphthalene), the substrate for melanin formation. We show that the DHN melanin pathway functions during N. crassa female development to generate melanized peridium and ascospore cell walls. N. crassa contains one polyketide synthase (PER-1), two polyketide hydrolases (PKH-1 and PKH-2), two THN (tetrahydroxynaphthalene) reductases (PKR-1 and PKR-2), and one scytalone dehydratase (SCY-1). We show that the PER-1, PKH-1, PKR-1 and SCY-1 are required for ascospoer melanization. We also identified the laccase that functions in the conversion of DHN into melanin via a free radical oxidative polymerization reaction, and have named the gene lacm-1 (laccase for melanin formation-1). In maturing perithecia, we show that LACM-1 is localized to the peridium cell wall space while the DHN pathway enzymes are localized to intracellular vesicles. We present a model for melanin formation in which melanin is formed within the cell wall space and the cell wall structure is similar to “reinforced concrete” with the cell wall glucan, chitin, and glycoproteins encased within the melanin polymer. This arrangement provides for a very strong and resilient cell wall and protects the glucan/chitin/glycoprotein matrix from digestion from enzymes and damage from free radicals.     

Purification and characterization of uricase,a nitrogen-regulated enzyme,from Neurospora crassa

Uricase, a purine catabolic enzyme, is controlled by induction and by nitrogen catabolite repression in Neurospora. Uricase was purified nearly 1000-fold to homogeneity. Only a single protein band could be detected in analytical gels of the pure enzyme, and the protein band in each case corresponded exactly to the position of in situ staining for enzyme activity. The molecular weight of native uricase was estimated to be 123,000 ± 7000. The enzyme is a tetramer composed of identical or similar-sized subunits. The K_m value of uricase for uric acid was estimated to be 4.2 × 10^?5, m. Oxonic acid was shown to be a competitive inhibitor of uricase, with a K_i value of 6.7 × 10^?7, m. Uricase is a stable enzyme and is not subject to feedback inhibition by ammonia, glutamate, or glutamine in Neurospora. The regulation of uricase appears to occur primarily at the biosynthesis level. Uricase appears to be a metalloenzyme with no essential sulfhydryl groups.     

Active transport of l-aspartic acid in Neurospora crassa

Transport of l-aspartic acid in Neurospora crassa conidia is shown to be mediated by neutral and general amino acid transport systems. The transport activity is dependent on pH and results in accumulation of l-aspartic acid against a gradient. Mutants deficient in transport of l-aspartic acid are described.     

Regulation of vectorial supply of vesicles to the hyphal tip determines thigmotropism in Neurospora crassa

Thigmotropism is the ability of an organism to respond to a topographical stimulus by altering its axis of growth. The thigmotropic response of the model fungus Neurospora crassa was quantified using microfabricated glass slides with ridges of defined height. We show that the polarity machinery at the hyphal tip plays a role in the thigmotropic response of N. crassa. Deletion of N. crassa genes encoding the formin, BNI-1, and the Rho-GTPase, CDC-42, an activator of BNI-1 in yeast, CDC-24, its guanine nucleotide exchange factor (GEF), and BEM-1, a scaffold protein in the same pathway, were all shown to significantly decrease the thigmotropic response. In contrast, deletion of genes encoding the cell end-marker protein, TEA-1, and KIP-1, the kinesin responsible for the localisation of TEA-1, significantly increased the thigmotropic response. These results suggest a mechanism of thigmotropism involving vesicle delivery to the hyphal tip via the actin cytoskeleton and microtubules. Neurospora crassa thigmotropic response differed subtly from that of Candida albicans where the stretch-activated calcium channel, Mid1, has been linked with thigmotropic behaviour. The MID-1 deficient mutant of N. crassa (Δmid-1) and the effects of calcium depletion were examined here but no change in the thigmotropic response was observed. However, SPRAY, a putative calcium channel protein, was shown to be required for N. crassa thigmotropism. We propose that the thigmotropic response is a result of changes in the polarity machinery at the hyphal tip which are thought to be downstream effects of calcium signalling pathways triggered by mechanical stress at the tip.     

Role of the redox state of nicoptinamide adenine dinucleotides in the in vivo regulation of inositol biosynthesis in Neurospora crassa

A possible _vivo role of pyridine nucleotides and their oxidized/reduced ratio on the regulation of inositol biosynthesis in Neurospora crassa was studied.A direct correlation was obtained when the values of of the water-soluble free inositol pool from intact N. crassa mycelia were plotted agains their NAD⁺/NADH or NAD⁺ + NADP⁺/NADH + NADPH ratios. Higher values in this inositol pool coincided with higher values in the chosen ratios.In long-term experiments (48 h), where the mold was grown without shaking, lower values for the inositol pool, the in vitro activity of D-glucose-6-phosphate cycloaldolase (glucocycloaldolase) and the myo-inositol (inositol) in phospholipids were found than those for cells grown with vigorous shaking.In short-time experiements (20 min), using N. crassa cells depleted of endogenous substrates, the in vivo synthesis of inositol was higher in cells incubated with vigorous shaking than in cells incubated without shaking. Nevertheless, in these experiments the in vitro activity of glucocycloaldolase was not affected by the earation conditions.