Hsp90-containing multiprotein complexes in the eukaryotic microbe Achlya

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.     

Biosynthesis of the oogoniols,steroidal sex hormones of Achlya: the role of fucosterol

Fucosterol-[3-³H] was converted to the oogoniols, sex hormones of Achlya, in A. heterosexualis. A similar conversion occurred in A. ambisexualis provided antheridiol was added.     

Metabolism of radioactive antheridiol by Achlya species

Antheridiol is a hormone produced by the water mold Achlya that induces the formation of male sex organs and the productionof oogoniol a second hormone that induces the formation of female sex organs. The fate of radioactive antheridiol in liquid cultures of heterothallic males and females of A. ambisexualis and A. bisexualis and the homothallics A. americana and A. conspicua was studied. After a lag period of 30–80 min many strains rapidly converted antheridiol to metabolite A. After a lag of 60 min a few strains converted metabolite A to metabolite B. The metabolites were at least a hundred times less active than antheridiol. From their R_f values they did not appear to be oogoniol. Cycloheximide and actinomycin D added before antheridiol or during the lag period prevented the formation of the metabolites except in the homothallic A. americana which already possessed the enzyme for the formation of metabolite A. Strains that readily acted as males were induced by antheridiol to metabolise antheridiol. Strong females were not induced by the antheriodiol concentrations used. It is suggested that metabolism of antheridiol could amplify the chemotropic stimulus for the male sex organs to grow up a gradient of antheridiol to the female organs.     

Detection of a pheromone-binding protein in the aquatic fungus Achlya ambisexualis

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 × 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.     

ULTRASTRUCTURAL AND ENZYMATIC EVIDENCE FOR THE PRESENCE OF MICROBODIES IN THE FUNGUS ACHLYA

Ultrastructural evidence for structures resembling microbodies is presented for the fungus Achlya ambisexualis Raper. These structures are DAB positive and thus presumably contain the enzyme catalase. Activities from mycelial homogenates for. the following enzymes are given: catalase, glycolate oxidase, uricase, isocitrate lyase, malate dehydrogenase, citrate synthetase, malate synthetase and glutamate: oxaloacetate transaminase. These results suggest that Achlya contains microbodies and that they may be of the glyoxysome type. The specific activity of catalase increases substantially following initiation of antheridial hyphae by the hormone antheridiol.     

Adenosylhomocysteine hydrolase inhibitors: synthesis of 5'-deoxy-5'-(isobutylthio)-3-deazaadenosine and its effect on Rous sarcoma virus and Gross murine leukemia virus.

One hour following administration of physiological concentrations of the steroid hormone antheridiol to a male strain of the water mold, $\text{Achlya}$$\text{ambisexualis}$, the rate of total cellular protein synthesis is increased. Further analysis revealed a sequential increase in the rate of syntheses for three classes of proteins following hormone stimulation. The rate of ribosomal protein synthesis increased as early as 20–30 minutes, followed by ribosomal salt wash proteins (40–60 minutes) and total soluble proteins after 60 minutes. Patterns of total cellular proteins, resolved by two-dimensional gel electrophoresis, during the first four hours after hormone treatment demonstrated the appearance of two newly synthesized peptides beginning at approximately 40 minutes followed by an increased rate of synthesis of three peptides after one hour. The synthesis of two peptides totally decreased after three hours of hormone induction.     

Antheridiol, RNA polymerase II and sexual development in the aquatic fungus, Achlya

The fungus, Achlya, is one of the most primitive eukaryotes known to secrete and respond to diffusible steroid sex hormones (pheromones). Antheridiol, which is produced by female strains of Achlya induces male strains to differentiate male sex organs. Induction of male strains with antheridiol elicits several changes in macromolecular synthesis including a quantitative enhancement in the synthesis of poly adenylated messenger RNA. We have examined whether this quantitative change is due to the regulation of RNA polymerase II. The level of polymerase increases as a response to the pheromone. This was examined using two different approaches, one of which included an extremely sensitive enzyme-linked immunosorbant assay (ELISA). Furthermore, the specific activities of polymerase II preparations isolated from pheromone-stimulated cultures was significantly higher than the specific activities of enzyme preparations isolated from control. A comparison of the polypeptide subunit composition of polymerase II preparations isolated from both pheromone-treated and control cultures on SDS polyacrylamide gels indicated no qualitative differences. Apparent differences in the stoichiometry of two specific subunits were reproducibly observed. The subunits of 140,000 and of 69,000 stained much more intensely in RNA polymerase II preparations isolated from 4h antheridiol-treated cultures.     

Nuclear envelope precursor vesicle targeting to chromatin is stimulated by protein phosphatase 1 in Xenopus egg extracts

The mechanism underlying targeting of the nuclear membrane to chromatin at the end of mitosis was studied using an in vitro cell-free system comprising Xenopus egg membrane and cytosol fractions, and sperm chromatin. The mitotic phase membrane, which was separated from a mitotic phase extract of Xenopus eggs and could not bind to chromatin, became able to bind to chromatin on pretreatment with a synthetic phase cytosol fraction of Xenopus eggs. When the cytosol fraction was depleted of protein phosphatase 1 (PP1) with anti-Xenopus PP1γ1 antibodies, this ability was lost. The addition of recombinant xPP1γ1 to the PP1-depleted cytosol fraction restored the ability. These and other results suggested that dephosphorylation of mitotic phosphorylation sites on membranes by PP1 in the synthetic phase cytosol fraction promoted targeting of the membranes to chromatin. On the other hand, a fragment containing the chromatin-binding domain of lamin B receptor (LBR) but not emerin inhibited targeting of membrane vesicles. It was also shown that PP1 dephosphorylates a phosphate group(s) responsible for regulation of the binding of LBR to chromatin. A possible mechanism involving PP1 and LBR for the regulation of nuclear membrane targeting to chromatin was discussed.     

Hormone-dependent lipoxygenase activity in Achlya ambisexualis

Homogenates of male Achyla ambisexualis oxidize exogenously added [¹⁴C]arachidonic acid to an unidentified lipoxygenase product. Synthesis occurs at a rate of 10.6 ± 1.3 μg mg⁻¹ protein 30 min⁻¹. Activity in homogenates of female mycelium is only 2.1 ± 1.2. Conversion is eliminated by the lipoxygenase inhibitor nordihydroguaiaretic acid (10⁻⁴ M). Homogenates prepared from the male grown in chemical but not physical contact with female mycelium had decreased lipoxygenase activity (3.1 ± 1.5), suggesting that antheridiol produced by the female decreases lipoxygenase activity in the male. To confirm this, actively growing male cultures were exposed to 10⁻⁹ M 7-deoxy-7-dihydroantheridiol, a stable analog of antheridiol, for 24 h. Homogenates from these cultures also had diminished lipoxygenase activity (2.7 ± 1.0). 7-Deoxy-7-dihydroantheridiol added to the incubation mixture at 10⁻⁹ M had no effect on lipoxygenase activity (9.0 ± 1.8), excluding a direct action of 7-deoxy-7-dihydroantheridiol on the enzyme. These data support the hypothesis that lipoxygenase products are associated with vegetative growth and suggest the antheridiol initiation of reproductive growth suppresses lipoxygenase activity.