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.     

Isolation and characterization of Neurospora crassa plasma membranes.

The isolation and characterization of plasma membranes from a cell wall-less mutant of Neurospora crassa are described. The plasma membranes are stabilized against fragmentation and vesiculation by treatment of intact cells with concanavalin A just prior to lysis. After lysis, the concanavalin A-stabilized plasma membrane ghosts are isolated by low speed centrifugation techniques and the purified ghosts subsequently converted to vesicles by removal of the bulk of the concanavalin A. The yield of ghosts is about 50% whereas the yield of vesicles is about 20%. The isolated plasma membrane vesicles have a characteristically high sterol to phospholipid ratio, Mg2+-dependent ATPase activity and (Na+ plus K+)-stimulated Mg2+ATPase activity. Only traces of succinate dehydrogenase and 5'-nucleotidase are present in the plasma membrane preparations.     

Isolation and characterization of plasma membranes from strains of Neurospora crassa with wild type morphology

A variety of commercially available cell wall hydrolytic enzyme preparations were screened alone and in various combinations for their ability to degrade the cell wall of Neurospora crassa wild type strain 1A. A combination was found which causes complete conversion of the normally filamentous germinated conidia to spherical structures in about 1.5 h. Examination of these spheroplasts by scanning electron microscopy indicated that, although they are spherical, they retain a smooth coat that can only be removed upon prolonged incubation in the enzyme mixture (about 10 h). The 10-h incubation in the enzyme mixture appears to have no obvious detrimental effects on the integrity of the plasma membrane since the activity and regulatory properties of the glucose active transport system in 10-h spheroplasts are essentially unimpaired. Importantly, plasma membranes can be isolated from the 10-h spheroplasts by an adaptation of the concanavalin A method developed previously in this laboratory for cells of the cell wall-less sl strain, which is not the case for the 1.5-h spheroplasts. The yield of plasma membrane vesicles isolated by this procedure is 18-36% as indicated by surface labeling with diazotized [125I]iodosulfanilic acid, and the preparation is less than 1% contaminated with mitochondrial protein. The chemical composition of the wild type plasma membranes is similar to that previously reported for membranes of the sl strain of Neurospora. The isolated wild type plasma membrane vesicles also exhibit all of the functional properties that have previously been demonstrated for the sl plasma membrane vesicles. The wild type vesicles catalyze MgATP-dependent electrogenic proton translocation as indicated by the concentrative uptake of [14C]SCN- and [14C]imidazole under the appropriate conditions, which indicates that they contain the plasma membrane H+-ATPase previously shown to exist in the sl plasma membranes and that they possess permeability barrier function as well. The vesicles also contain a Ca2+/H+ antiporter as evidenced by their ability to catalyze protonophore-inhibited MgATP-dependent 45Ca2+ accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of the isolated vesicles indicate that the protein composition of the wild type vesicles is roughly similar to that of the sl plasma membranes with the H+-ATPase present as a major band of Mr approximately 105,000. The wild type plasma membrane ATPase forms a phosphorylated intermediate similar to that of the sl ATPase, and the specific activity of the H+-ATPase in both wild type and sl membranes is approximately 3 mumol of Pi released/mg of protein/min.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of Neurospora crassa plasma membrane ATPase.

A variety of the biochemical properties of the electrogenic plasma membrane ATPase of Neurospora crassa are described. The enzyme catalyzes the hydrolysis of ATP, resulting in the formation of ADP and inorganic phosphate. Optimal activity is observed between pH 6 and 6.5. ATP hydrolysis approaches a maximum rate at an Mg-ATP concentration of 10–20 mm with a half-maximal velocity around 2 mm Mg-ATP. The enzyme requires a divalent cation for activity in the following order of preference at 10 mm: Mg²⁺, Co²⁺ > Mn²⁺ > Zn²⁺ > Fe²⁺, Ca²⁺, Cu²⁺. The enzyme is quite specific for ATP compared to the other nucleotides tested. Treatment of the plasma membranes with sodium deoxycholate inactivates the ATPase and the inactivation can be prevented by the addition of certain acidic phospholipids with the deoxycholate. Other classes of lipids cannot prevent the deoxycholate inhibition. The organic mercurials parachloromercuribenzoate and parachloromercuriphenylsulfonate are potent inhibitors of the ATPase, but N-ethylmaleimide at a similar concentration is not inhibitory. The organic mercurial inhibition is not reversed by mercaptoethanol. Under appropriate conditions, the inhibitory effect of p-chloromercuribenzoate is suppressed in the presence of ATP. Treatment of the plasma membranes with trypsin leads to a marked inhibition of the ATPase activity and this inhibition can be prevented by Mg-ATP. Neither the organic mercurial reactive site(s) nor the trypsin-sensitive site(s) are accessible from the outer surface of the plasma membranes. Some of the implications of the above findings are discussed.     

Identification and properties of an ATPase in vacuolar membranes of Neurospora crassa

Using a vacuolar preparation virtually free of contamination by other organelles, we isolated vacuolar membranes and demonstrated that they contain an ATPase. Sucrose density gradient profiles of vacuolar membranes show a single peak of ATPase activity at a density of 1.11 g/cm3. Comparison of this enzyme with the two well-studied proton-pumping ATPases of Neurospora plasma membranes and mitochondria shows that it is clearly distinct. The vacuolar membrane ATPase is insensitive to the inhibitors oligomycin, azide, and vanadate, but sensitive to N,N'-dicyclohexylcarbodiimide (Ki = 2 microM). It has a pH optimum of 7.5, requires a divalent cation (Mg2+ or Mn2+) for activity, and is remarkably unaffected (+/- 20%) by a number of monovalent cations, anions, and buffers. In its substrate affinity (Km for ATP = 0.2 mM), substrate preference (ATP greater than GTP, ITP greater than UTP greater than CTP), and loss of activity with repeated 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid washes, the vacuolar membrane ATPase resembles the F1F0 type of ATPase found in mitochondria and differs from the integral membrane type of ATPase in plasma membranes.     

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.     

Permeability changes in membranes of Neurospora crassa after freezing and thawing.

Conidia of Neurospora crassa which are in different physiological states show different rates of survival after freezing and thawing. [¹⁴C]adenine uptake by frozen and thawed conidia in different physiological states show a correlation with their survival. The uptake method was extended to study the survival of mycelium in log phase and stationary phase. From the uptake data it appears that log phase mycelium is extremely sensitive to all rates of freezing and thawing studied, while the stationary phase mycelium showed slight tolerance to freezing, if freezing was done at a slow rate. A study of the efflux of labeled compounds from the conidia in various physiological states or from the mycelia after freezing and thawing showed that, although efflux followed the same general trend as survival in conidia, it did not relate to the survival in mycelium, suggesting that the death of conidia or mycelium in the freeze-thaw treatments is not due to efflux of compounds.     

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.     

Characterization of plasma membrane adenosine triphosphatase of Neurospora crassa.

It has been proposed (Slayman, C.L., Long W.S., and Lu, C.Y.-H. (1973) J. Membr. Biol. 14, 305--338) that in Neurospora crassa, a plasma membrane ATPase functions to pump H+ ions out of the cell, thereby generating an electrochemical gradient that can drive transport processes. Using the concanavalin A method of Scarborough (Scarborough G.A. (1975)J. Biol. Chem. 250, 1106--1111), we have prepared plasma membranes of Neurospora and have deomonstrated that they do contain a distinct ATPase activity with the following properties. It has a pH optimum of 6.0, is highly specific for ATP (hydrolyzing other nucleoside triphosphates less than 6% as rapidly), requires Mg2+ at concentrations approximately equimolar to the concentration of ATP, is weakly stimulated by certain monovalent cations (K+ and NH4+) and anions (SCN- and acetate), is inhibited by N,N'-dicyclohexylcarbodiimide, but is not affected by oligomycin or ouabain. The plasma membrane fraction also contains residual mitochondrial contamination, which can be determined quantitatively by assaying oligomycin-sensitive ATP-ase activity, at pH 8.25, and succinic dehydrogenase activity.     

Cyclic AMP and the plasma membrane potential in Neurospora crassa.

Diverse treatments, which have been shown by Slayman, C. L. (1977) in Water Relations in Membrane Transport in Plants and Animals (Jungreis, A., Hodges, T. K., Kleinzeller, A., and Schultz, S. G., eds) pp. 69-86, Academic Press, New York, to depolarize the plasma membrane of Neurospora, increase levels of adenosine 3':5'-monophosphate (cyclic AMP) in the organism. The treatments include those producing large transport fluxes of metabolizable or nonmetabolizable compounds, rapid temperature drops, and addition of agents which uncouple oxidative phosphorylation. Severe mechanical stress, which may also act to depolarize the plasma membrane, leads to increases in cyclic AMP. The maximal depolarization appears to precede the maximal cyclic AMP levels. It is proposed that the membrane depolarization produces the increased cyclic AMP levels by stimulating the plasma membrane-bound adenylate cyclase and that cyclic AMP may be important to the maintenance of membrane integrity.     

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.     

The effects of branched chain fatty acid incorporation into Neurospora crassa membranes

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), an unusual branched chain fatty acid thought to disrupt the hydrophobic regions of membranes, can be incorporated into the lipids of growing Neurospora cultures. The phytanic acid must be supplied in a water soluble form, esterified to a Tween detergent (Tween-Phytanic). This fatty acid and its oxidation product, pristanic acid, were found in both the phospholipid and neutral lipid fractions of Neurospora. In phospholipids of the wild-type strain, phytanic acid was present to the extent of 4 to 5 moles percent of the fatty acids and pristanic acid, about 41 moles percent. The neutral lipids contained 42 and 4 moles percent of phytanic and pristanic acids respectively. By employing a fatty acid-requiring mutant strain (cel^?), the phytanic acid level was raised to a maximum of 16 moles percent in the phospholipids and to 63 moles percent in the neutral lipids. Under this condition, the level of pristanic acid was reduced to about 6 moles percent in phospholipids and 1 mole percent in the neutral lipids. The phytanic acid levels could not be further elevated by increased supplementation with phytanic acid or by a change in the growth temperature. In strains with a high phytanic acid content, the complete fatty acid distribution of the phospholipids and neutral lipids was determined. In the neutral lipids, phytanic acid appeared to replace the 18 carbon fatty acids, particularly linoleic acid. The presence of phytanic acid in the phospholipids was confirmed by mass spectrometry, and by the isolation of a phospholipid fraction containing this fatty acid via silicic acid column chromatography. Most of the phytanic acid in phospholipids appeared to be in phosphatidylethanolamine, and 2 lines of evidence suggest that it was esterified to both positions of this molecule. In the fatty acid-requiring mutant strain (cel^?), the replacement by phytanic acid of 10 to 15% of the fatty acids in the phospholipid produced an aberrant morphological change in the growth pattern of Neurospora and caused this organism to be osmotically more fragile than the wild-type strain. The lack of noticeable effect of the high levels of pristanic acid in the phospholipids suggests that it is not just the presence of the methyl groups in a branched chain fatty acid which leads to the altered membrane function in this organism.     

Purification and characterization of the plasma membrane ATPase of Neurospora crassa

The plasma membrane of Neurospora crassa contains a proton-translocating ATPase, which functions to generate a large membrane potential and thereby to drive a variety of H+-dependent co-transport systems. We have purified this ATPase by a three-step procedure in which 1) loosely bound membrane proteins are removed by treatment with 0.1% deoxycholate; 2) the ATPase is solubilized with 0.6% deoxycholate in the presence of 45% glycerol; and 3) the solubilized enzyme is purified by centrifugation through a glycerol gradient. This procedure typically yields approximately 30% of the starting ATPase activity in a nearly homogeneous enzyme preparation of high specific activity, 61-98 mumol/min/mg of protein. The membrane-bound and purified forms of the ATPase are very similar with respect to kinetic properties (pH optimum, nucleotide and divalent cation specificity, sigmoid dependence upon Mg-ATP concentration) and sensitivity to inhibitors (including N,N'-dicyclohexylcarbodiimide and vanadate). Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified ATPase displays a single major polypeptide band of Mr = 104,000, which is essentially identical in its electrophoretic mobility with the large subunit of [Na+, K+]-ATPase of animal cell membranes and [Ca2+]-ATPase of sarcoplasmic reticulum. The structural similarity of the fungal and animal cell ATPases, together with the fact that both are known to form acyl phosphate intermediates, suggests that they may share a common reaction mechanism.     

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.     

Rubidium transport in Neurospora crassa

Rb⁺ transport in low-K⁺ cells of Neurospora crassa is biphasic, transport at millimolar Rb⁺ being added to a transport process which saturates in the micromolar range. Both processes exhibit Michaelis-Menten kinetics, but in the micromolar phase the kinetic parameters depend on the K⁺ content of the cell (the lower the K⁺ content the lower the K_m and the higher the V_max). Normal-K⁺ cells, suspended in a buffer with millimolar K⁺, do not present Rb⁺ transport in the micromolar range. Millimolar transport in these cells presents kinetics which depend on the K⁺ in buffer (the higher the K⁺ the higher the K_m), although the K⁺ content of the cells is constant. Na⁺ inhibits competitively Rb⁺ transport in low-K⁺ and normal-K⁺ cells, but, even when the differences between the Rb⁺K_m values are more than three orders of magnitude, the apparent dissociation constant for Na⁺ is the same, and millimolar, in both cases.