Glycogen synthesis in the fungus Neurospora crassa

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [¹⁴C]glucose from ADP-[¹⁴C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose: glycogen glucosyltransferase as compared with those of the already known UDP glucose: glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-[¹⁴C]glucose or ADP-[¹⁴C]glucose released all the radioactivity as maltose after α or β amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[¹⁴C]glucose was the substrate but the stimulation was much greater with ADP-[¹⁴C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent on the presence of a ‘primer’ of the α 1 → 4 glucan type.     

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.     

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.     

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.     

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.     

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.     

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.     

Aspects of the carbohydrate metabolism of a mutant of Neurospora crassa requiring acetate for growth

1.

1. Two mutant genes controlling the activities of different enzyme systems in Neurospora are described. One controls the activity of the enzyme pyruvic carboxylase, the other an enzyme system involved in the oxidation of pyruvic acid.     

Characterization of a 45-kDa polypeptide as the precursor of subunit 1 of cytochrome c oxidase in Neurospora crassa

In a previous paper (Van 't Sant, P., Mak, J.F.C. and Kroon, A.M. (1981) Eur. J. Biochem. 121, 21–26) we showed the existence of three elongated precursor proteins (45, 36 and 25 kDa) of mitochondrial translation products in Neurospora crassa. We presented some indications that the largest precursor could be related to subunit 1 of cytochrome c oxidase. Here we present conclusive evidence that the 45-kDa polypeptide is indeed this precursor by demonstrating that an immunodetectable 45-kDa polypeptide displays the same behaviour as the labeled 45-kDa precursor; both accumulate after long incubation with cycloheximide or by decreasing the temperature and both are not tightly membrane bound. Moreover the antibody against subunit 1 of cytochrome c oxidase also recognizes, in immunoadsorption experiments, besides subunit 1, the 45-kDa polypeptide accumulated by cycloheximide incubation. Furthermore, we developed a small scale purification of antibodies against subunit 1 of cytochrome c oxidase. By means of these purified antibodies it is demonstrated that the 45-kDa polypeptide and subunit 1 have corresponding antigenic determinants. Under the various conditions tested, all three precursors are less firmly membrane-bound than the mature subunits. Finally, it is observed that in short incubations in vivo, chloramphenicol inhibits the processing of the mitochondrially synthesized precursors, under conditions where mitochondrial translation is only partially inhibited.     

The functional relationship between the polymerization and catalytic activity of beef liver glutamate dehydrogenase. III. Analysis of Thusius' critique.

Thusius' critique (1977) of our work (Cohen et al., 1975,1976; Cohen &; Benedek, 1976), on the functional relationship between the equilibrium polymerization and catalytic activity of beef liver glutamate dehydrogenase, is refuted on a point by point basis. Thusius' critique is primarily based on data relating to the kinetics of the polymerization-depolymerization reaction. It is shown that such data are not in contradiction to our equilibrium thermodynamic analysis, for this analysis makes absolutely no predictions regarding the kinetics of the polymerization-depolymerization reaction. Moreover, the important functional relationship between the polymerization and allosteric control of beef liver glutamate dehydrogenase has, in any event, been clearly demonstrated on a purely experimental basis. Furthermore, our theoretical model is the only proposed model capable of quantitatively explaining the available data on the detailed equilibrium polymer distribution and the associated level of catalytic activity as functions of effector and enzyme concentrations.     

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.     

Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers.

The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure from pairs of purified variants. Several chromatographically distinct peaks of differing enzymological properties were purified from each hybridization mixture in quantities of up to a few milligrams, and represented distinct species of hybrid hexamers differing in subunit ratio.     

Isolation and characterization of a mutant of Neurospora crassa deficient in general amino acid permease activity

A mutant of Neurospora crassa (pm-nbg²⁷) was isolated on the basis of its resistance to p-fluoro-phenylalamine on ammonium-deficient Vogel's medium. This mutant was found to be devoid of both conidial and post-conidial (after 180 min of preincubation) transport acitvity of all amino acids.Genetic analysis exhibiting of pm-nbg²⁷ by crossing it to wild-type (74^A) resulted in the predicted segregants exhibiting transport characteristics of pm-n, pm-b, pm-g, pm-nb, pm-ng, pm-bg and parental types.The above observations confirm the postulated general amino acids permease system as well as a single genetic locus control of that activity.