Plant and fungus calmodulins are polyubiquitinated at a single site in a Ca2(+)-dependent manner

In plants Ca2+ plays a crucial role as second messenger. Thus calmodulin is one of the most important signal transducing molecules for metabolic regulation in plants. Previously we showed that bovine testis calmodulin can be covalently coupled at one site to ubiquitin in a Ca2(+)-dependent manner in the presence of ATP/Mg2+ by ubiquityl-calmodulin synthetase. Since calmodulin from spinach has 13 amino acid sequence differences to bovine calmodulin - two of them in Ca2(+)-binding loops - it was unclear, whether a conjugation of ubiquitin to this molecule would be possible. In this paper it is shown that calmodulin from spinach and a similar calmodulin from the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca2(+)-dependent manner. It is shown that higher molecular mass conjugates containing up to three ubiquitin molecules per calmodulin are obtained. Experiments with methylated ubiquitin demonstrate that, as with vertebrate calmodulins, only one lysine residue is linked to ubiquitin and that the incorporation of additional ubiquitin molecules leads to a polyubiquitin chain.     

An enzymatic assay for calmodulins based on plant NAD kinase activity

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K0.5's ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K0.5's for the activation of Ca2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca2+ (10% of maximum), but no effect in the absence of Ca2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures.     

Activation of Neurospora crassa soluble adenylate cyclase by calmodulin.

The soluble form of adenylate cyclase was extracted and purified from wild-type Neurospora crassa mycelia. Brain or N. crassa calmodulin significantly enhanced this enzyme activity in assay mixtures containing Mg2+-ATP as substrate. EGTA reverses this calmodulin activation.     

Characterization of Neurospora crassa cyclic AMP phosphodiesterase activated by calmodulin.

Activation of cyclic AMP phosphodiesterase I by brain or Neurospora calmodulin was studied. The stimulation required micromolar concentrations of Ca2+, and it was observed at cyclic AMP concentrations between 0.1 and 500 microM. Activation was blocked by EDTA and some neuroleptic drugs such as chlorpromazine and fluphenazine. These drugs inhibit the elongation of N. crassa wild-type aerial hyphae. These results reinforce the evidence towards the recognition of Ca2+-calmodulin as one of the systems controlling cyclic nucleotide concentrations in Neurospora.     

Production and specificity of monoclonal antibodies against calmodulin from Dictyostelium discoideum

Monoclonal antibodies were raised against calmodulin purified from Dictyostelium discoideum. To increase its antigenicity, the calmodulin was conjugated to keyhole limpet hemocyanin; mice were immunized with the conjugate. Hybridomas producing antibodies against calmodulin were identified by screening culture supernatants with calmodulin coupled to bovine serum albumin. The specificity of antibodies from hybridoma culture supernatants was tested by Western blot of Dictyostelium cell lysates. For the purpose, methods were developed that permitted sensitive detection of calmodulin bound to membranes. The key elements of the blotting protocol were used of PVDF membrane, transfer conducted in phosphate buffer, and glutaraldehyde fixation after transfer. These methods permitted detection of as little as 0.1 ng of calmodulin spotted directly onto the membrane, or 10 ng transferred from an SDS polyacrylamide gel. Ten calmodulin-specific antibodies were identified; most of these reacted preferentially with the calcium-containing form of Dictyostelium calmodulin. Several of the monoclonal antibodies cross-reacted with calmodulin from bovine brain.     

Ca(2+)-dependent ubiquitination of calmodulin in yeast.

Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner by ubiquityl-calmodulin synthetase (uCaM-synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253-256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM-synthetase conjugated ubiquitin Ca(2+)-dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (reticulocyte) uCaM-synthetase. As estimated from autoradiograms the monoubiquitination product (first-order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23-26 kDa and the second-order conjugate an apparent molecular mass of ca. 28-32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.     

Calmodulin from neurospora crassa. General properties and conformational changes

Calmodulin from Neurospora crassa has been purified to electrophoretic homogeneity. Equilibrium gel filtration experiments suggest that its Ca-binding properties are indistinguishable from those of vertebrate calmodulins. The isoelectric point of 4.04 and electrophoretic behavior under nondenaturing conditions indicate that N. crassa calmodulin is slightly less acidic than its vertebrate counterpart. The amino acid composition is typical of plant calmodulins with the exception that trimethyllysine is absent and that the content of Ser is unusually high. The tryptic peptide map of N. crassa calmodulin reveals an important number of point mutations as compared to vertebrate calmodulin. Differences in primary structure may explain why N. crassa calmodulin is less potent in the activation of myosin light chain kinase than calmodulins from higher organisms. The far UV circular dichroic spectra of the Ca-, Mg-, and metal-free forms of N. crassa calmodulin are similar to those of vertebrate calmodulin; in contrast, the near UV circular dichroic spectra are very different, apparently due to the differences in Tyr content. The single Tyr residue of N. crassa calmodulin, presumably located in position 138, undergoes an inversion of optical chirality upon addition of Ca2+, but not of Mg2+, to the metal-free protein.     

Ubiquitination of endogenous calmodulin in rabbit tissue extracts.

Previously we were able to show that purified calmodulins from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner. It was therefore pertinent to answer the question if a tissue extract contains all the components necessary for the endogenous synthesis of ubiquityl calmodulin (uCaM). Therefore [125I]ubiquitin, ATP/Mg2+ and Ca2+ were added to tissue extracts enriched by a single ion exchange step. In such extracts of red blood cells, skeletal muscle and testis a novel ubiquitin conjugate of 27-29 kDa is formed. This novel band could be identified as ubiquityl-calmodulin by the following methods: (i) identical Rf-value of novel conjugate and standard uCaM in SDS-PAGE; (ii) Ca(2+)-dependent conjugate formation; (iii) Ca(2+)-dependent adsorption to fluphenazine-Sepharose; (iv) Ca(2+)-dependent mobility change of the novel conjugate during SDS-PAGE; and (v) inhibition of conjugate band formation by phosphorylase kinase. These experiments clearly demonstrate that ubiquityl calmodulin can be endogenously generated in enriched cellular extracts and strongly indicate that this reaction is of importance in vivo.     

Rapid purification of calmodulin and S-100 protein by affinity chromatography with melittin immobilized to sepharose

Melittin-Sepharose was prepared for Ca2+-dependent affinity chromatography of calmodulin and S-100 protein. This matrix exhibits extremely high capacity (approximately 10 mg calmodulin/ml gel), low nonspecific binding, and excellent recovery (greater than 90%) under optimal conditions. Recovery of calmodulin from melittin-Sepharose was related to the degree of saturation of column capacity with lower yields when only partial saturation was achieved. Large-scale, simultaneous purification of calmodulin and S-100 protein from brain was carried out using selective adsorption to organomercurial agarose followed by melittin-Sepharose chromatography; yields were 250-300 mg of calmodulin and 200-300 mg of S-100 per kg tissue. Calmodulin also was purified in a single step from bovine testis supernatant using melittin-Sepharose in yields comparable to those from brain.     

Immunocharacterization of actin and its immunofluorescent localization during the developmental gametophytic stages of Allomyces arbuscula

Highly specific polyclonal antibodies against actin from Allomyces arbuscula were produced in rabbits, immunopurified by immunoblotting and specified with actin isolated from Neurospora crassa and mouse skeletal muscle. Used as immunofluorescence probes, they allowed localization of actin in the sequential gametophytic stages of the mould.     

Optimized vectors and selection for transformation of Neurospora crassa and Aspergillus nidulans to bleomycin and phleomycin resistance.

To provide a dominant selectable marker for transformation of Neurospora crassa strains lacking specific auxotrophic mutations, we have engineered the bleomycin (Bm) resistance-encoding gene (ble) from the bacterial transposon Tn5 for expression in N. crassa. The coding region of the ble gene was fused to the promoter and terminator regions of the N. crassa am gene. In some vectors, multiple cloning sites were placed flanking the ble gene to provide a versatile ble cassette. When introduced into N. crassa, the hybrid ble gene conferred resistance to greater than 15 micrograms Bm/ml. Under optimal conditions, the levels of Bm required (2.5 micrograms/ml) make even large-scale transformation experiments very economical. Aspergillus nidulans could also be efficiently transformed to Bm resistance using the N. crassa ble gene fusion. Since the ble gene functions in both N. crassa and A. nidulans, the gene should be useful as a transformation marker for the many other filamentous fungi which are sensitive to Bm.     

Purification of a phosphatidylinositol/phosphatidylcholine transfer protein from Neurospora crassa.

This paper reports, for the first time, the purification of a phospholipid transfer protein (PLTP) from a fungus, Neurospora crassa. The protein was purified from the post-microsomal supernatant of N. crassa by successive chromatography on DEAE-cellulose, Sephadex-G75 and PBE 94 (pH 4-7). The purified protein (M(r) 38,000) was found to transfer phosphatidylinositol preferentially over phosphatidylcholine, like the PLTP from the yeast, Saccharomyces cerevisiae. PC transfer was completely inhibited by inactivation of free amino groups or tryptophan residues. Surprisingly, the protein did not cross-react with antibodies against the bovine brain PITP. The cellular content of the protein was maximal during the logarithmic phase of growth. However, no direct correlation between the content of the protein and PC transfer activity could be demonstrated.     

Resistance to Azole Drugs in Neurospora crassa

Staben, C. 1995. Resistance to azole drugs in Neurospora crassa. Experimental Mycology 19: 163-165. Neurospora crassa was susceptible to azole drugs: ketoconazole (MIC 1 μg/ml), fluconazole (MIC 5 μg/ml), and SCH39304 (MIC 5 μg/ml). Mutants of N. crassa resistant to ketoconazole were selected and genetically characterized. The seven characterized resistance mutations represented at least four genetic loci. Some mutants, but not all, were also resistant to fluconazole and to SCH39304.     

Quantification of the secretory glycoproteins of the subcommissural organ by a sensitive sandwich ELISA with a polyclonal antibody and a set of monoclonal antibodies against the bovine Reissner’s fiber

The subcommissural organ (SCO) is an ependymal brain gland that releases glycoproteins into the ventricular cerebrospinal fluid where they condense to form the Reissner’s fiber (RF). We have developed a highly sensitive and specific two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of the bovine SCO secretory material. The assay was based on the use of the IgG fraction of a polyclonal antiserum against the bovine RF as capture antibody and a pool of three peroxidase-labeled monoclonal antibodies that recognize non-overlapping epitopes of the RF glycoproteins as detection antibody. The detection limit was 1 ng/ml and the working range extended from 1 to 4000 ng/ml. The calibration curve, generated with RF glycoproteins, showed two linear segments: one of low sensitivity, ranging from 1 to 125 ng/ml, and the other of high sensitivity between 125 and 4000 ng/ml. This assay was highly reproducible (mean intra- and interassay coefficient of variation 2.2% and 5.3%, respectively) and its detectability and sensitivity were higher than those of ELISAs using exclusively either polyclonal or monoclonal antibodies against RF glycoproteins. The assay succeeded in detecting and measuring secretory material in crude extracts of bovine SCO, culture medium supernatant of SCO explants and incubation medium of bovine RF; however, soluble secretory material was not detected in bovine cerebrospinal fluid.     

Isolation and characterization of the adenylate cyclase structural gene of Neurospora crassa.

A single gene (nac) encoding an adenylate cyclase was cloned from the genomic DNA library of Neurospora crassa, using the DNA fragment encoding the catalytic domain of adenylate cyclase of Saccharomyces cerevisiae as a probe. The open reading frame of this gene (6900 base pairs) was interrupted three time by introns. The protein encoded consists of 2300 amino acids and has adenylate cyclase activity. N. crassa adenylate cyclase has a high degree of homology with the catalytic domains of yeast and bovine brain adenylate cyclases.     

Purification of a 47-kDa calmodulin-binding polypeptide as an actin-binding protein from Neurospora crassa

We have enriched a 47-kDa polypeptide (p47) from Neurospora crassa on the basis of its affinity to calmodulin. The p47 was purified to homogeneity by chromatography on a Mono S cation exchange column and evidence is presented that the polypeptide co-sediments specifically with F-actin. The intracellular distribution of p47 and actin was also examined using indirect double immunofluorescence staining of cells at different stages of development. Our results suggest that by altering the conformation binding site of actin to p47, calmodulin could play a regulatory role in the polarized hyphal growth of N. crassa.     

Control of nucleotide and erythroascorbic acid pools by cyclic AMP in Neurospora crassa

UDPglucuronic acid and erythroascorbic acid were identified in extracts of the fungus Neurospora crassa. The concentrations of these two compounds are estimated, in growing wild type N. crassa, to be about 0.10 and 0.28 mumol/ml of cell water, respectively. The pools of these two compounds are regulated by cyclic AMP in Neurospora, both being elevated in the cr-1, adenylate cyclase deficient mutant and both being lowered by exogenous cyclic AMP. The pools of these two compounds are also elevated on nitrogen deprivation. The pools of a large number of other nucleotides are not influenced by cyclic AMP. Possible relationships between the metabolism of UDPglucuronic acid and erythroascorbic acid are discussed. It was found that exogenous cyclic AMP was much more effective in influencing cultures grown at 30-37 degrees C than those grown at 25 degrees C. We suggest that higher temperatures may render Neurospora more permeable to a variety of different compounds.     

Calcium-binding phosphoprotein: the principal acidic protein of mammalian sperm

A calcium-binding phosphoprotein previously found only in brain and adrenal medulla has been isolated from the adult bovine testis. The testicular protein is electrophoretically indistinguishable from the protein of adult bovine brain and adrenal medulla in 15% polyacrylamide gels at pH 8.3 and 4.7. Crude homogenates and acidic protein fractions of adult testis, fetal testis and epididymal spermatozoa were examined electrophoretically for the presence of this calcium-binding protein. The protein was present in homogenates of adult testis and epididymal spermatozoa but only to limited extent in the homogenate of fetal testis. It was the major acidic protein of spermatozoa. It appears likely that the calcium-binding protein evident in adult testicular tissue is contributed largely by the developing spermatozoa.     

Phylogenetic conservation of epitopes in mammalian aldose reductase: application to immunoquantitation

Rabbit antibodies raised against bovine kidney aldose reductase (ALR2) were shown to be monospecific by Western blot analysis of kidney homogenates. In addition, the antiserum (alpha-BKALR2) reacts with a single electrophoretic species in homogenates from rabbit, porcine, and human kidney. ALR2 has been detected in homogenates of bovine kidney, heart, brain and lens, and estimation of the enzyme level in these tissues was accomplished by densitometric analysis of Western blots. Standard curves using highly purified bovine kidney ALR2 were linear in the range of 5-100 ng; a similar sensitivity was seen in tissue homogenates. The results presented here for the ALR2 level in bovine tissues (kidney greater than heart greater than brain greater than lens) are in agreement with literature values for those tissues from which the enzyme has previously been purified. The interspecies similarity in electrophoretic mobility and the retention of antibody reactivity suggest extensive phylogenetic epitope conservation in mammalian aldose reductase.     

Isolation of complex III from various mitochondria. Comparison of the structural and functional properties of the preparations from beef heart, calf liver and Neurospora crassa

Active complex III was isolated by an improved procedure from beef heart mitochondria, from Neurospora crassa mitochondria and for the first time from mitochondria originating from mammalian tissue other than heart, i.e. calf liver. The described procedure consists of differential extraction of the respective mitochondria, hydroxyapatite chromatography and, finally, either gel- or affinity chromatography. The preparations contain the well known prosthetic groups, i.e. 6-8 mumol b-type heme, 3-4 mumol c-type heme and 5-8 mumol non-heme iron per g of protein. The preparations from beef heart and from calf liver mitochondria are indistinguishable in their subunit composition by sodium dodecyl sulfate polyacrylamide gel electrophoresis, whereas the preparation from Neurospora crassa mitochondria is clearly different. The phospholipid content of all preparations is rather low, amounting to about 100 mumol/g protein. The molar catalytic activity of ubiquinol-9-cytochrome c reductase at 25 degrees C amounts to 50s-1 for the N. crassa complex III and 70-100s-1 for the bovine complexes. After reincorporation into phospholipid vesicles, all preparations how tight coupling between electron transfer from ubiquinol to cytochrome c and proton translocation across the phospholipid bilayer.