Two-dimensional electrophoresis of plasma membranes, showing differences among wild-type and abnormal ascospore mutant strains of Neurospora crassa.

Plasma membranes isolated from vegetative cultures of wild-type Neurospora crassa were analyzed by two-dimensional electrophoresis, followed by staining with silver nitrate to visualize proteins and fluorescein-labeled concanavalin A to visualize glycosylated subunits. Mycelial plasma membranes from strains carrying mutations affecting ascospores were also analyzed. Two of the mutant strains were shown to have aberrant two-dimensional membrane subunit patterns. The correlation of these abnormalities with the known electron microscopic evidence for aberrations of their ascospore-delimiting membrane during ascospore genesis is discussed.     

Permeability measurements on mitochondria from wild-type and poky strains of Neurospora crassa.

The permeability properties of isolated Neurospora mitochondria were determined by measuring the rate at which the mitochondria swell in isotonic solutions of various organic and inorganic molecules. Like mammalian mitochondria, wild-type Neurospora mitochondria were impermeable to sucrose and only slightly more permeable to most inorganic ions (K, Na, Cl). Their permeability to K was greatly increased by valinomycin and by monensin. In addition, the mitochondria contain specific systems mediating PO4 uptake and PO4- malate, fumarate, and succinate exchange. Mitochondria from the maternally inherited poky strain of Neurospora, previously demonstrated to possess defective ribosomes and a grossly cytochrome chain, showed a slight but significant increase in permeability to inorganic ions. They contained, however, the specific uptake and exchange systems for phosphate and dicarboxylate anions, a result suggesting that these systems do not depend upon mitochondrially synthesized polypeptides.     

Energy-linked potassium uptake by mitochondria from wild-type and poky strains of Neurospora crassa.

Mitochondria from Neurospora crassa, like mammalian mitochondria, carry out rapid, energy-linked K+ uptake and H+ release in the presence of valinomycin. The maximal rate of K+ uptake was about 1.0 mumol/mg of mitochondrial protein per min and was seen at valinomycin concentrations in the range of 100 to 200 mug per mg of mitochondrial protein and at K+ concentrations of 4 mM or above. Uptake could be supported either by substrate oxidation or by adenosine 5'-triphosphate (ATP), and was inhibited in the former case by antimycin or cyanide, in the latter case by oligomycin, and in both cases by 2,4-dinitrophenol. Mitochondria from the cytochrome-deficient mutant poky carried out substrate-driven K+ uptake at reduced rates, but oligomycin-sensitive, ATP-driven K+ uptake at rates about 60% greater than those shown by wild-type mitochondria. This result is consistent with the recent finding (Mainzer and Slayman 1976) that poky contains elevated amounts of oligomycin-sensitive mitochondrial adenosine 5'-triphosphatase activity.     

Asexual development is increased in Neurospora crassa cat-3-null mutant strains

We use asexual development of Neurospora crassa as a model system with which to determine the causes of cell differentiation. Air exposure of a mycelial mat induces hyphal adhesion, and adherent hyphae grow aerial hyphae that, in turn, form conidia. Previous work indicated the development of a hyperoxidant state at the start of these morphogenetic transitions and a large increase in catalase activity during conidiation. Catalase 3 (CAT-3) increases at the end of exponential growth and is induced by different stress conditions. Here we analyzed the effects of cat-3-null strains on growth and asexual development. The lack of CAT-3 was not compensated by other catalases, even under oxidative stress conditions, and cat-3^RIP colonies were sensitive to H₂O₂, indicating that wild-type (Wt) resistance to external H₂O₂ was due to CAT-3. cat-3^RIP colonies grown in the dark produced high levels of carotenes as a consequence of oxidative stress. Light exacerbated oxidative stress and further increased carotene synthesis. In the cat-3^RIP mutant strain, increased aeration in liquid cultures led to increased hyphal adhesion and protein oxidation. Compared to the Wt, the cat-3^RIP mutant strain produced six times more aerial hyphae and conidia in air-exposed mycelial mats, as a result of longer and more densely packed aerial hyphae. Protein oxidation in colonies was threefold higher and showed more aerial hyphae and conidia in mutant strains than did the Wt. Results indicate that oxidative stress due to lack of CAT-3 induces carotene synthesis, hyphal adhesion, and more aerial hyphae and conidia.

     

Adenosine kinase-deficient mutant of Neurospora crassa.

Tubercidin-resistant mutant strains of Neurospora crassa were isolated, and at least one appeared to be deficient in adenosine kinase. No significant differences in [8-14C]adenosine labeling of purine nucleotides or nucleosides were found between the wild type and the adenosine kinase-deficient strains.     

A perithecial color mutant of Neurospora crassa

Summary A mutation, per-1, was found in N. crassa which caused orange instead of black perithecia when the protoperithecial parent contained the per-1 allele. No effect of per-1 was found on vegetative morphology or nutritional requirements. The per-1 locus was mapped proximal to iv (?6201) in the right arm of linkage group V.     

Electrical phenotypes of calcium transport mutant strains of a filamentous fungus, Neurospora crassa

We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters-a mechanosensitive channel homolog (MscS), a Ca(2+)/H(+) exchange protein (cax), and Ca(2+)-ATPases (nca-1, nca-2, nca-3)-as well as those of double mutants (the nca-2 cax, nca-2 nca-3, and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2-containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H(+)-ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca(2+) levels, indicative of lesions in Ca(2+) homeostasis. However, the net Ca(2+) effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca(2+)-selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca(2+) signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca(2+)] was elevated. Thus, although Ca(2+) homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654-661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H(+)-ATPase activity.     

Mitochondrial adenosine triphosphatase of wild-type and poky Neurospora crassa.

We have compared the adenosine triphosphatase (ATPase) activity of mitochondria prepared from wild-type Neurospora crassa and from poky, a maternally inherited mutant known to possess defective mitochondrial ribosomes and reduced amounts of cytochromes aa3 and b. poky contains two distinct forms of mitochondrial ATPase. The first is normal in its Km for ATP, specificity for nucleotides and divalent cations, pH optimum, cold stability, and sensitivity to inhibitors (oligomycin, N,N-dicyclohexyl carbodiimide, and adenylyl imidodiphosphate). The fact that membrane-bound, cold-stable, oligomycin-sensitive ATPase activity is present in poky (with an activity of 1.93 +/- 0.03 mumol/min-mg of protein compared with 1.33 +/- 0.07 mumol/min-mg of protein in the wild-type strain) and also in chloramphenicol-grown wild-type cells suggests that products of mitochondrial protein synthesis play only a limited role in the attachment of the mitochondrial ATPase to the membrane in Neurospora. poky also contains a second form of mitochondrial ATPase, which has an activity of 1.5 +/- 0.2 mumol/min-mg of protein, is oligomycin sensitive but cold labile, and presumably is attached less firmly to the mitochondrial membrane. The two forms, added together, represent a substantial overproduction of mitochondrial ATPase by poky.     

Letter: Methylation of mitochondrial RNA species in the wild-type and poky strains of Neurospora crassa.

Methylation of mitochondrial RNAs in the me-3 and poky f⁺ me-3 strains of Neurospora crassa has been re-examined using procedures based on steadystate labeling with [methyl-³H]methionine and taking the precaution of adding sodium formate to suppress randomization of the methyl-³H label. Under these conditions, the values measured for the mitochondrial ribosomal RNAs are 0·05 to 0·16 methyl group per 100 nucleotides, much lower than had been reported previously and, in addition, there is no longer a significant difference between the me-3 and poky f⁺ me-3 strains (contrast Kuriyama &; Luck, 1974). Control experiments rule out the possibility that the much lower values result from grossly inefficient labeling of intra-mitochondrial methyl groups.     

Neurospora crassa mutant impaired in glutamine regulation.

The final products of the catabolism of arginine that can be utilized as nitrogen sources by Neurospora crassa are ammonium, glutamic acid, and glutamine. Of these compounds, only glutamine represses arginase and glutamine synthetase. We report here the isolation and characterization of a mutant of N. crassa whose arginase, glutamine synthetase, and amino acid accumulations are resistant to glutamine repression (glnI). This mutant has a greater capacity than the wild type (glns) to accumulate most of the arginine and some of the glutamine in osmotically sensitive compartments while growing exponentially. Nonetheless, the major part of the glutamine remains soluble and metabolically available for repression. We propose that the lower repression of glutamine synthetase by glutamine in this mutant could be a necessary condition for sustaining the higher flow of nitrogen for the accumulation of amino acids observed in ammonium excess and that, if glutamine is the nitrogen signal that regulates the arginine accumulation of the vesicle, the glnr mutant has also escaped this control. Finally, in the glnr mutant, some glutamine resynthesis is necessary for arginine biosynthesis and accumulation.