phytosterols and antheridiol

The chaperone Hsp90 plays a key role in the maturation and activation of many 'client' proteins in eukaryotic cells. In the oomycete Achlya ambisexualis two populations of hsp90 transcripts that differ slightly in size (2.8 and 2.9 kb) are present in heat-shocked mycelia. Only the 2.8 kb transcripts are seen in vegetative mycelia and in mycelia undergoing antheridiol-induced differentiation. Two different hsp90 cDNAs were isolated and characterized. Although nearly identical, an additional eight nucleotide sequence was present at the end of the 3'UTR of one of the two cDNAs. RT-PCR analyses indicated that hsp90 transcripts containing the eight nucleotide extension, were present only in heat-shocked mycelia. Hsp90 transcripts lacking this sequence were present in vegetative mycelia and the levels of these transcripts increased in both heat-shocked and hormone-treated mycelia. Each hsp90 cDNA encoded a nearly identical Hsp90 protein. However, two Hsp90 proteins (86 and 84 kDa) were observed on immunoblots of mycelial proteins. Only one of these, i.e., the 86 kDa protein was detected by an anti-phosphoserine antibody, suggesting that the difference in mass of the two Hsp90 isoforms, was due at least in part, to different levels of phosphoserine residues.

Topics: 3' Untranslated Regions; Achlya; Algal Proteins; Amino Acid Motifs; Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA, Complementary; Gene Expression Regulation; HSP90 Heat-Shock Proteins; Molecular Sequence Data; Mycelium; Nucleic Acid Conformation; Phosphorylation; Phosphoserine; Phytosterols; RNA, Messenger; Sequence Alignment; Sequence Analysis, DNA; Temperature

In the oomycete fungus Achlya ambisexualis, hyphae of the male strain undergo sexual differentiation in the presence of the steroid hormone antheridiol. Earlier studies demonstrated that antheridiol binds with high affinity to a 9S multiprotein complex from A. ambisexualis cytosols. Although these complexes were found to contain the heat shock protein Hsp90, the other components were not known. It was of interest to determine if any of the other protein components in the Achlya Hsp90-heterocomplexes would be homologous to those found in the steroid receptor-Hsp90-heterocomplexes of vertebrates. Cytosolic proteins of 110 kDa, 74 kDa, 64 kDa, 61 kDa, 56 kDa, 47 kDa, 27 kDa and 23 kDa, were found in repeated trials, to co-immunoprecipitate with Achlya Hsp90. The 74 kDa protein was identified as the heat shock protein Hsp70, the 23 kDa protein was found to be related to the vertebrate protein p23 and the 56 kDa protein was found to be related to immunophilin FKBP51. All three of these proteins are components of the vertebrate receptor heterocomplexes. The 110 kDa, 61 kDa and 27 kDa proteins appeared to be unique to the Achlya complexes. Unlike the seven other proteins co-immunoprecipitating with Hsp90, the 61 kDa protein was observed only in the co-immunoprecipitates produced from in vitro translates of RNA isolated from antheridiol-treated mycelia.

Topics: Animals; Antibodies, Fungal; Antibodies, Monoclonal; Carrier Proteins; Cell-Free System; Cytosol; DNA-Binding Proteins; Fungal Proteins; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Macromolecular Substances; Multiprotein Complexes; Oomycetes; Phytosterols; Precipitin Tests; Rabbits; RNA, Fungal; Tacrolimus Binding Proteins

When mycelia of Achlya ambisexualis J. Raper strain E87 were undergoing antheridial branching, a marked increase was observed in the levels of transcript populations encoding the heat shock protein chaperone Hsp90 and transcript populations encoding three different Hsp70-family heat shock protein chaperones, respectively. Although up to 90% of hyphae in the hormone-treated thalli were undergoing antheridial branching, no similar increase in the level of transcripts encoding actin was observed. Nuclear run-on assays demonstrated that the observed antheridiol-induced increases in the levels of the chaperone RNAs resulted from increased transcription. Although not tested for function, the nucleotide sequence of the 5' flanking region of each of the two A. ambisexualis hsp90 genes revealed a diversity of sequences and motifs similar or identical to the sequences of known transcription factor response elements. Among these potential response element sequences observed in the A. ambisexualis genes were motifs observed also in animal steroid hormone response elements. Surrounding the primer-extension determined transcription start site of each A. ambisexualis hsp90 gene was a 16-nucleotide sequence that matched in 14 out of 16 nucleotides a sequence found in the transcription initiation region of many different oomycete genes.

Topics: Base Sequence; Cell Nucleus; DNA Primers; Gene Expression Regulation, Fungal; Hot Temperature; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Molecular Sequence Data; Oomycetes; Phytosterols; Sequence Alignment; Sequence Homology, Nucleic Acid; Transcription, Genetic

In the filamentous oomycete fungus Achlya, the differentiation of gamete bearing structures on vegetative hyphae of the male mating type, is induced by the Achlya steroid hormone, antheridiol. Among the several metabolically labeled intracellular proteins whose synthesis or accumulation is altered by hormone treatment are steroid-induced 85-kDa and 68- to 78-kDa proteins. The 85-kDa protein was previously shown to be the Achlya heat shock protein hsp85 [Brunt et al., 1990; Brunt and Silver, 1991], a component of the putative Achlya steroid hormone receptor. It was of interest to determine if the antheridiol-induced "70-kDa" proteins were hsp70-family heat shock proteins and if hormone treatment-induced changes in the level of hsp70 transcripts. Two different Achlya hsp70 genomic sequences were cloned and used to investigate these questions. The two hsp70 sequences recognized two different mycelial transcript populations, one of which was regulated also by decreased glucose. Of note, both of the two hsp70 transcript populations were found to be regulated by antheridiol. The hormone-induced changes in hsp70 transcript levels were temporally correlated with the onset of massive lateral hyphal branching and alterations in the pattern of secreted N-linked glycoproteins which occur in hormone-treated mycelia. To our knowledge, this represents one of the first reports on changes in hsp70 proteins and transcripts during fungal differentiation. Our results may have implications for the role of heat shock proteins in hyphal branching and secretion in filamentous fungi and perhaps other cell types.

Topics: Blotting, Northern; Blotting, Southern; Cell Differentiation; DNA, Fungal; Gene Expression Regulation, Fungal; Heat-Shock Proteins; Oomycetes; Phytosterols; Restriction Mapping; Sex Differentiation; Transcription, Genetic

In the fungus Achlya, sexual development in the male strain E87 is mediated by the steroid hormone antheridiol. Treatment of vegetatively growing cells of E87 with antheridiol resulted in changes in the [35S]methionine labeling of several secreted proteins. The most heavily labeled group of proteins observed in the secreted fraction from control cells appeared on one-dimensional gels as a prominent wide band which could be resolved into three closely spaced components with relative molecular weights (MWs) of 57, 54, and 50 kD, respectively. After hormone treatment the two lower MW proteins of the group were barely detectable. Concomitant with the observed reductions in the labeling of the 54 and 50 kD proteins was the increased labeling of a doublet of very prominent proteins with relative MWs of 44.4 and 43 kD, respectively. The results of experiments with Endoglycosidase H suggested that the 44.4 and 43 kD proteins seen in hormone-treated cultures, might result from the removal or reduced synthesis of high mannose oligosaccharide groups found on the 54 and 50 kD proteins normally seen in control cultures. Additional support for this suggestion was provided by the observation that the 54 and 50 kD proteins from control cultures appeared to bind to conA columns and to be eluted with alpha-methylmannoside, while there was little or no binding of the 44.4 and 43 kD proteins from hormone-treated cells. Although other possibilities are not excluded, the results are suggestive of a steroid hormone-induced alteration in glycoprotein processing. The functions of the observed hormonally-responsive secreted proteins as well as those of the secreted proteins in non-hormone-treated cultures, are not known at this time.

Topics: Acetylglucosaminidase; Chytridiomycota; Electrophoresis, Polyacrylamide Gel; Fungal Proteins; Glycoproteins; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Molecular Weight; Oomycetes; Phytosterols; Tunicamycin

In the fungus Achlya ambisexualis sexual development in the male strain E87 is controlled by the steroid hormone antheridiol. To investigate the effects of antheridiol on the synthesis and/or accumulation of specific cellular proteins we have analysed [35S]methionine-labeled proteins from control and hormone-treated cells using both one-dimensional (1D) and two-dimensional (2D) PAGE. Since in a total cell extract, hormone-induced changes in specific proteins might not be apparent against a background of more abundant proteins, cells were fractionated prior to protein isolation. It was also necessary to establish a concentration of hormone carrier, in this case methanol, which by itself did not alter the pattern of protein synthesis. Using these approaches the addition of the hormone antheridiol to vegetatively growing cells of Achlya E87 was found to result in changes in the synthesis and/or accumulation of at least 16 specific proteins, which could be localized to the cytoplasmic, nuclear or cell wall/cell membrane fractions. The most prominent changes observed in the hormone-treated cells included the appearance in the cytoplasmic fraction of labeled proteins at 28.4 and 24.3 kD which were not detectable in control cells, and a significant enrichment in the labeling of a 24.3 kD protein in the cell wall/cell membrane fraction. A marked increase in the labeling of 85, 63 and 47 kD proteins in the nuclear fraction from hormone-treated cells was also noted. The molecular weight (MW) and the behavior on 2D gels of the 85 kD hormone-induced protein appeared very similar to that of the 85 kD heat-shock protein reported in Achlya. Quantitive changes in the [35S]methionine labeling of several other proteins were noted in all three cell fractions.

Topics: Cell Compartmentation; Cell Membrane; Cell Nucleus; Cell Wall; Chytridiomycota; Cytoplasm; Fungal Proteins; Molecular Weight; Oomycetes; Phytosterols; Time Factors

Sexual reproduction in the eukaryotic fungi Achlya is controlled by two steroid pheromones. Antheridiol is the steroid released by female cells that induces male sexual differentiation. The antheridiol-induced response of male cells has been shown to be influenced by the composition of the culture medium. The present study was designed to determine if the composition of the culture media might also affect the levels of antheridiol binding protein in the cytosol of male cells. The mycelial content of cytosolic steroid pheromone binding sites in Achlya ambisexualis E87 males was measured at daily intervals during 6 days of suspension culture in media containing different nitrogen sources. Levels of binding sits increased during the first 2 days in culture to a plateau that was maintained for the next 2-3 days. During the first 3 days in culture, levels were much lower in mycelia cultured in an enriched medium containing lactalbumin hydrolysate compared to mycelia cultured in defined media containing glutamic acid as the nitrogen source. The level of binding sites increased rapidly when mycelia were transferred from an enriched medium to a nutrient-free salt solution and decreased when mycelia were transferred from a defined to an enriched medium. The relative differences in cytosolic binding measured by in vitro radioligand saturation analysis were confirmed by in vivo uptake studies. It is concluded that the mycelial content of antheridiol binding sites can be experimentally manipulated by variations in the composition of the culture medium and/or the time period of incubation in the medium.

Topics: Binding Sites; Carrier Proteins; Chytridiomycota; Culture Media; Oomycetes; Phytosterols; Tritium

We have previously reported the discovery of a specific high-affinity binding protein for the fungal sex steroid pheromone antheridiol in the cytosol of Achlya ambisexualis male cells. In this report, we describe the fractionation of the binding protein from the cytosol by ammonium sulfate precipitation, the optimization of in vitro conditions for radioligand binding assays, and some of the biochemical properties of the binding protein. In the presence of sodium molybdate, the macromolecule has a sedimentation coefficient of 8.3 S in sucrose gradients of low ionic strength, a Stokes radius of 56.6 A (Sephacryl S-300 columns), a molecular weight of approximately 192,000, a frictional ratio of 1.5, and an axial ratio of 8.9. The binding protein can be eluted with 0.24 M KCl as a single peak from DEAE-Sephadex A-25 columns. These results indicate that this steroid-binding protein from a primitive eukaryotic microbe has in vitro biochemical properties that are similar to those of other known steroid receptors in higher organisms.

Topics: Cytosol; Fungi; Kinetics; Molecular Weight; Phytosterols; Protein Conformation; Receptors, Steroid

Sexual reproduction among the eukaryotic fungi of the genus, Achlya, is controlled by two steroid pheromones. Antheridiol is the pheromone constitutively produced by female cells that induces male sexual differentiation and development. A biologically active tritium-labeled derivative of antheridiol, [1,2-3H]7-deoxy-7-dihydro-antheridiol ( [3H]7dA), has been synthesized. Radioligand-binding studies have revealed the presence of a specific binding protein in the cytosol of male cells that may represent the endogenous receptor for antheridiol. Binding to this macromolecule was characterized by an apparent equilibrium dissociation constant and maximum binding capacity of approx. 7 X 10(-10) M and 1 100-2 000 fmoles/mg protein, respectively. Sedimentation analysis in sucrose gradients revealed that the binding protein distributes in the 8S region under low ionic strength and sodium molybdate-stabilized conditions. Under conditions of high ionic strength, in the presence or absence of 10 mM sodium molybdate, the binding site distributes in the 3.6S region of the gradient. Analysis of radioligand binding in the presence of other steroids and steroid hormones revealed that the binding is specific for antheridiol and its analog.

Topics: Centrifugation, Density Gradient; Chemoreceptor Cells; Cytoplasm; Fungi; Phytosterols; Protein Binding

Topics: Binding Sites; Chromatin; DNA-Directed RNA Polymerases; Fungi; Oomycetes; Phytosterols; Rifampin; RNA; Templates, Genetic; Transcription, Genetic

Topics: Fungal Proteins; Fungi; Oomycetes; Phytosterols; Time Factors