The separation and properties of two phosphoprotein phosphatases from the dimorphic fungus Mucor rouxii

Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I (M_r 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K_0.5 for MnCl₂ of 2 mm. Enzyme II (M_r 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn²⁺. This enzyme was strongly inhibited by 50 mm NaF or 1 mm ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn²⁺, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn²⁺. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.     

Two different intrachain cAMP sites in the cAMP-dependent protein kinase of the dimorphic fungus Mucor rouxii

cAMP sites of the cAMP-dependent protein kinase from the fungus Mucor rouxii have been characterized through the study of the effects of cAMP and of cAMP analogs on the phosphotransferase activity and through binding kinetics. The tetrameric holoenzyme, which contains two regulatory (R) and two catalytic (C) subunits, exhibited positive cooperativity in activation by cAMP, suggesting multiple cAMP-binding sites. Several other results indicated that the Mucor kinase contained two different cooperative cAMP-binding sites on each R subunit, with properties similar to those of the mammalian cAMP-dependent protein kinase. Under optimum binding conditions, the [3H]cAMP dissociation behavior indicated equal amounts of two components which had dissociation rate constants of 0.09 min-1 (site 1) and 0.90 min-1 (site 2) at 30 degrees C. Two cAMP-binding sites could also be distinguished by C-8 cAMP analogs (site-1-selective) and C-6 cAMP analogs (site-2-selective); combinations of site-1- and site-2-selective analogs were synergistic in protein kinase activation. The two different cooperative binding sites were probably located on the same R subunit, since the proteolytically derived dimeric form of the enzyme, which contained one R and one C component, retained the salient properties of the untreated tetrameric enzyme. Unlike any of the mammalian cyclic-nucleotide-dependent isozymes described thus far, the Mucor kinase was much more potently activated by C-6 cAMP analogs than by C-8 cAMP analogs. In the ternary complex formed by the native Mucor tetramer and cAMP, only the two sites 1 contained bound cAMP, a feature which has also not yet been demonstrated for the mammalian cAMP-dependent protein kinase.     

The presence of two heat-stable inhibitors of phosphoprotein phosphatases in the dimorphic fungus, Mucor rouxii

Two heat-stable inhibitors (a and b) of phosphoprotein phosphatases I and II from Mucor rouxii were isolated from mycelium of the fungus. They were partially purified from extracts by heating, DEAE-cellulose chromatography, and Sephadex G-75 gel filtration. The molecular weights of inhibitors a and b, estimated by gel filtration, are 5,000 and 20,000 respectively. Inhibitor a acts similarly on both enzymes while inhibitor b is relatively more active on enzyme II. Storage of inhibitor b at -20 degrees C for several weeks resulted in a partial conversion to a lower-molecular-weight form with properties similar to those of inhibitor a.     

DNA methylation and polyamines in regulation of development of the fungus Mucor rouxii.

DNA from intact or spherically growing spores of Mucor rouxii is highly methylated, whereas DNA from germlings has low levels of methylation. DNA from spores incubated with hydroxyurea or 1,4-diaminobutanone is also highly methylated. The reversal of the effect of 1,4-diaminobutanone by azacytidine correlated with DNA hypomethylation. These data suggest that the change in growth pattern from spherical to polarized correlates with the degree of DNA methylation and that this, in turn, may be controlled by polyamine levels.     

Two distinct classes of polyuronide from the cell walls of a dimorphic fungus, Mucor rouxii

Polyuronides were extracted from purified yeast and mycelial walls of Mucor rouxii by sequential treatments with lithium chloride and potassium hydroxide and were fractionated by ion-exchange chromatography on DEAE-Sephadex. Two polymers (I and II) of different acidity were found in both wall types. Polymer I contained D-glucuronic acid, L-fucose, D-mannose, and much smaller amounts of D-galactose. Yeast and mycelial polymer I had similar uronic acid contents but differed in their neutral sugar compositions and molecular weights. Polymer II from both cell types contained largely D-glucuronic acid and had similar molecular weights. On partial acid hydrolysis, both polymers I and II gave rise to insoluble glucuronans which appeared to be homopolymeric. One-third of the total uronosyl residues of polymer I, and almost all of the uronosyl residues of polymer II, were present in homopolymeric segments. However, homopolymers derived from polymers I and II may not be identical.     

Polymeric structure of the cyclic AMP-dependent protein kinase from the dimorphic fungus Mucor rouxii and purification of its catalytic subunit

Summary The polymeric structure of the cyclic AMP-dependent protein kinase (E.C.2.7.1.37) from the dimorphic fungus Mucor rouxii was analyzed through studies of gel filtration and sucrose gradient centrifugation of the holoenzyme and its subunits and by photoaffinity labeling of the regulatory subunit. It was demonstrated that it is a tetramer composed by two regulatory subunits (R) of mol. wt. 75 000 and two catalytic subunits (C) of mol. wt. 41 000 forming a holoenzyme R₂C₂ of mol. wt. 242 000. Frictional coefficients of 1.55 and 1.62 for the holoenzyme and for the regulatory dimer, respectively, indicate a significant degree of dimensional asymmetry in both molecules. A procedure for the purification of the catalytic subunit of the kinase is presented. The holoenzyme could be bound to a cyclic AMP-agarose column and the catalytic subunit could be eluted by 0.5 M NaCl, well resolved from the bulk of protein. This particular behaviour of the holoenzyme in cyclic AMP-agarose chromatography allowed the inclusion of this step in the purification of the catalytic subunit and corroborated that the holoenzyme was not dissociated by cyclic AMP alone. The isolated catalytic subunit displays Michaelis-Menten behaviour towards kemptide, protamine and histone and is inhibited by sulfhydryl reagents, indicating that the molecule has at least one cysteine residue essential for enzyme activity. The catalytic activity of the isolated C subunit is inactivated by the mammalian protein kinase inhibitor, and is inhibited by the regulatory subunit from homologous and heterologous sources. In general, the properties of the catalytic subunit suggest a structural similarity between Mucor and mammalian C subunits.Abbreviations C catalytic subunit monomer of protein kinase - R regulatory subunit monomer of protein kinase - 8-N₃-cyclic AMP 8-azido-cylic AMP - SDS sodium dodecyl sulfate - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) See AcknowledgementsCareer Investigators from the CONICET     

Further studies on cyclic adenosine 3':5'-monophosphate protein kinase from dimorphic fungus Mucor rouxii

In this paper, cyclic adenosine-3′:5′-monophosphate-dependent protein kinase from yeast-like cells of Mucor rouxii is characterized. A scheme of partial purification is described together with K_m for ATP (15 μm), histone (0.2 mg/ml), half-maximal activation constant for cyclic AMP (30 nm), and dissociation constant for the binding of cyclic AMP (40 nm). This enzyme is similar to type II protein kinases in two main aspects: the elution position in DEAE-cellulose chromatography and the readiness of its reassociation. But it has a singular characteristic: it does not dissociate completely with cyclic AMP alone (even at concentrations as high as 0.3 mm) unless histone or NaCl is present. NaCl displays several roles: helps dissociation, prevents inactivation of the catalytic subunit, inhibits enzyme activity, and does not prevent reassociation as occurs with type II protein kinases. Once the holoenzyme is dissociated, cyclic AMP is essential to maintain the enzyme in the dissociated state.     

Studies on cyclic adenosine 3' ,5'-monophosphate levels, Adenylate cyclase and phosphodiesterase activities in the dimorphic fungus Mucor rouxii.

The germination of spores of Mucor rouxii into hyphae was inhibited by 2 mm dibutyryl cyclic adenosine 3′,5′-monophosphate or 7 mm cyclic adenosine 3′,5′-monophosphate; under these conditions spores developed into budding spherical cells instead of filaments, provided that glucose was present in the culture medium. Removal of the cyclic nucleotides resulted in the conversion of yeast cells into hyphae. Dibutyryl cyclic adenosine 3′,5′-monophosphate (2 mm) also inhibited the transformation of yeast to mycelia after exposure of yeast culture to air.Since in all living systems so far studied adenylate cyclase and cyclic adenosine 3′,5′-monophosphate phosphodiesterase are involved in maintaining the intracellular cyclic adenosine monophosphate level, the activity of both enzymes and the intracellular concentration of cyclic adenosine monophosphate were investigated in yeast and mycelium extracts. Cyclic adenosine monophosphate phosphodiesterase and adenylate cyclase activities could be demonstrated in extracts of M. rouxii. The specific activity of adenylate cyclase did not vary appreciably with the fungus morphology. On the contrary, cyclic adenosine monophosphate phosphodiesterase activity was four- to sixfold higher in mycelial extracts than in yeast extracts and reflected quite accurately the observed changes in intracellular cyclic adenosine monophosphate levels; these were three to four times higher in yeast cells than in mycelium.     

Actin in Mucor rouxii

Rhodamine-conjugated phalloidin was used to analyze the actin distribution during hyphal formation in Mucor rouxii. The occurrence of actin patches in the cortical region of the cells was seen in the initial stages of growth. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-sepharose. This peptide was identified on immunoblots when polyclonal antibodies against rabbit muscle actin were used as a probe. These results indicate: (1) that changes in actin localization accompany the hyphal development and (2) the fungal 43 kDa protein shares properties that are common to muscle actin.     

Actin in Mucor rouxii

Abstract Rhodamine-conjugated phalloidin was used to analyze the actin distribution during hyphal formation in Mucor rouxii . The occurrence of actin patches in the cortical region of the cells was seen in the initial stages of growth. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-sepharose. This peptide was identified on immunoblots when polyclonal antibodies against rabbit muscle actin were used as a probe. These results indicate: (1) that changes in actin localization accompany the hyphal development and (2) the fungal 43 kDa protein shares properties that are common to muscle actin.     

Function and regulation of the acid and neutral trehalases of Mucor rouxii

Two different trehalose-hydrolysing activities, known as acid or non-regulatory trehalases, and neutral or regulatory trehalases, have been recognised in a number of fungal species. The true role of these apparently redundant hydrolases remained obscure for many years. However, recent evidence suggests that neutral trehalases would be specialised in the mobilisation of cytosolic trehalose, while acid trehalases would only hydrolyse extracellular trehalose. Results obtained with Mucor rouxii, a Zygomycete initially thought to posses only neutral trehalase activity, reinforced this hypothesis. M. rouxii grows efficiently in trehalose as the sole carbon source. Trehalose-grown or carbon-starved cells exhibit a high trehalase activity of optimum pH 4.5, bound to the external surface of the cell wall, in contrast with the neutral (pH 6.5) trehalase, which occurs in the cytosol. Other differences between the neutral and the acid trehalases are the temperature optimum (35°C and 45°C, respectively) and thermal stability (half-life of 2.5 min and 12 min at 45°C, respectively). The neutral trehalase, but not the acid trehalase, is activated in vitro by cAMP-dependent phosphorylation, stimulated by Ca²⁺, and inhibited by EDTA. It shows maximal activity at germination and decreases as growth proceeds. In contrast the activity of the acid trehalase is totally repressed in glucose-grown cultures and increases upon exhaustion of the carbon source, and is strongly induced by extracellular trehalose.     

Sporangiospore lipids of the mycelial fungus Mucor ramannianus incapable of dimorphic growth

Members of the species Mucor ramannianus are believed to be monomorphic. They grow only as a mycelium and are not capable of growth as budding cells, i.e., of dimorphic growth. In our study, we investigated the lipid composition of M. ramannianus sporangiospores, which retained the capability of initiating mycelial growth in the course of long-term cultivation of the spore-forming mycelium. It was demonstrated that sporangiospores contained high concentrations of triacylglycerides (TAG) in their reserve lipids and high concentrations of phosphatidylcholine (PC) in their membrane lipids; low concentrations of methylated ergosterol precursors were detected among sterols. On the basis of the data presented, in order to evaluate the potential of mucor fungi for yeastlike growth, it has been suggested to analyze the qualitative and quantitative characteristics of their sporangiospore lipids and to consider the following criteria as the criteria of sporangiospore capacity for giving rise to yeastlike growth upon spore germination: (1) the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio; (2) the level of ergosterol and the ratio between the methylated and demethylated sterols; as well as (3) the ratios between phospholipids and glycolipids (PL/GL) and (4) between etherified and free sterols (ES/FS).     

Multiple protein kinase activities in the dimorphic fungus Mucor rouxii. Comparison with a cyclic adenosine 3',5'-monophosphate binding protein.

Protein kinase and cyclic adenosine 3′,5′-monophosphate (cAMP) binding activities have been detected in cell extracts of the dimorphic fungus Mucor rouxii. The subcellular distribution of both activities indicates that most of the binding protein is in the high-speed supernatant (S₁₀₀), while about 70% of the total protein kinase activity remains in particulate fractions. S₁₀₀ preparations have been analyzed by diethylaminoethyl cellulose column chromatography. Binding activity can be resolved in two peaks (A and B) and protein kinase in three peaks (I, II, and III). Peaks I and II are casein dependent and insensitive to cAMP. Peak III utilizes histone as substrate and is activated (two- to fourfold) by cAMP. Theophylline strongly inhibits cAMP binding activity and mimics the effect of cAMP on cAMP-dependent protein kinase. The possible relationship between cAMP binding activity and cAMP-dependent protein kinase is suggested.     

Calmodulin-like activity in Mucor rouxii

Abstract A modulator factor with properties similar to those of calmodulin was found and partially purified from the soluble fraction of Mucor rouxii . These properties include: heat-stability, stimulation in a Ca²⁺-dependent fashion of cyclic nucleotide phosphodiesterase from bovine brain, and inhibition of this process by trifluoperazine. This calmodulin-like activity was detected in the soluble fraction of both mycelium and yeast-like cells of the fungus.     

Chitosanases in the autolysis of Mucor rouxii

The mycelium of Mucor rouxii reached a 50% degree of lysis after 50 days incubation, and was then stable with the incubation time. The pH of the medium was 4.3 when autolysis began, rising to pH 7.6 after 6 days of autolysis and remaining there for the duration of the experiment. Maximum degradation of mycelium occurs during the first days of autolysis. Glucosamine is present in the culture liquid during all the autolytic process. Enzymes implicated in the degradation of chitosan and chitin were studied in the culture fluid during autolysis. An exochitosanase activity was detected after a day of autolysis, and its activity increased during 20 days of autolysis and afterwards remained constant until the end of the process. An endochitosanase activity was detected in the culture fluid from the beginning of the autolysis, having its maximum activity after 34 days of incubation. Both activities show an optimum pH of 5.5, but the pH range of activity for endochitosanase was broader than for exochitosanase. Both activities were not inhibited by 0.5 mM glucosamine. Activities of the enzymes B-N-acetylglucosaminidase and chitinase were not found. The chitosan content in the cell walls decreased with the incubation time. In these cell walls the chitin content experienced an increase at the beginning of the autolysis, decreasing afterwards. The enzymatic complex obtained from autolyzed cultures of M. rouxii hydrolyzed 2-day-old cell walls of this fungus. The hydrolysis was 21% after 24 h of incubation, liberating glucose and glucosamine. As a consequence protoplasts from M. rouxii germinated spores were obtained with its own lytic enzymes in adequate osmotic conditions. The involvement of chitosanases in the autolysis of this fungus have been studied.     

Control of dimorphism in Mucor rouxii

Haidle, C. W. (The University of Texas, Austin), and R. Storck. Control of dimorphism in Mucor rouxii. J. Bacteriol. 92:1236-1244. 1966.-Yeastlike cells of Mucor rouxii NRRL 1894 were converted to filaments in a medium containing glucose, mineral salts, casein hydrolysate, nicotinic acid, and thiamine when the gas phase was changed from CO(2)-N(2) or N(2) alone to air. Germ tubes began to appear 3 to 4 hr after exposure to air. Ribonucleic acid (RNA) precursors were incorporated into RNA in a discontinuous fashion during this conversion, but the incorporation was continuous during the anaerobic growth of yeastlike cells and during the aerobic germination of sporangiospores. The incorporation of labeled amino acids during the conversion was exponential. Labeling of ribosomal RNA occurred as shortly as 5 min after replacement of CO(2)-N(2) with air. However, P(32)-labeled RNA isolated 20 min after exposure to air had a guanine plus cytosine (GC) content of 41% (mole%) as compared with the 47% found for labeled and unlabeled RNA isolated at other stages of the life cycle of this organism or later during the conversion. In addition, the overall base composition of this 20-min pulse-labeled RNA resembled that of deoxyribonucleic acid (GC = 39%), suggesting that a significant proportion of this RNA is of the messenger type. Furthermore, the synthesis of cytochrome oxidase was induced upon exposure of yeastlike cells to air. Cyanide, acriflavine, and cycloheximide, which inhibited the action or synthesis of cytochrome oxidase, also inhibited the yeast to filament transition.     

Regulation of ornithine decarboxylase activity in Mucor bacilliformis and Mucor rouxii

Ornithine decarboxylase (ODC) from Mucor bacilliformis and Mucor rouxii was studied. Enzymatic activity was maximal at pH 7.2–7.4 and at 30°C. The K_m was 0.17 mM for the M. bacilliformis enzyme. Putrescine was a competitive inhibitor of ODC with a K_i of 2–3 mM. Enzymatic activity was undetectable in sporangiospores but increased rapidly during the first stages of spore swelling, reaching the highest levels during germ tube or bud emergence, and then decreased. Incubation at 30°C inhibited spore germination in M. bacilliformis and prevented development of ODC activity. More ODC activity was present in mycelial than in yeast cells. Morphological transition of yeast cells into hyphae by an anaerobic-aerobic shift induced a rapid and transient increase in ODC activity. Similar results were obtained when the morphogenetic transformation of M. rouxii was induced by CO₂ elimination in an anaerobic environment. Transfer of mycelial cells to anaerobiosis resulted in a rapid decrease in enzyme activity. Changes in ODC activity were accompanied by a change in the pool of polyamines. The possible role of ODC in growth and cell differentiation in Mucor is discussed.