RESUMO
In Neurospora crassa, caffeine and other methylxanthines are known to inhibit phosphodiesterase (PDE) activity, leading to augmented cAMP levels. In this organism, it has also been shown that the addition of these drugs significantly lengthens the circadian period, as seen by conidiation rhythms. Utilizing in vivo bioluminescence reporters, pharmacological inhibitors, and cAMP analogs, we revisited the effect of methylxanthines and the role of cAMP signaling in the Neurospora clockworks. We observed that caffeine, like all tested methylxanthines, led to significant period lengthening, visualized with both core-clock transcriptional and translational reporters. Remarkably, this phenotype is still observed when phosphodiesterase (PDE) activity is genetically or chemically (via 3-isobutyl-1-methylxanthine) abrogated. Likewise, methylxanthines still exert a period effect in several cAMP signaling pathway mutants, including adenylate cyclase (cr-1) and protein kinase A (PKA) (Δpkac-1) mutants, suggesting that these drugs lead to circadian phenotypes through mechanisms different from the canonical PDE-cAMP-PKA signaling axis. Thus, this study highlights the strong impact of methylxanthines on circadian period in Neurospora, albeit the exact mechanisms somehow remain elusive. IMPORTANCE Evidence from diverse organisms show that caffeine causes changes in the circadian clock, causing period lengthening. The fungus Neurospora crassa is no exception; here, several methylxanthines such as caffeine, theophylline, and aminophylline cause period lengthening in a concentration-dependent manner. Although methylxanthines are expected to inhibit phosphodiesterase activity, we were able to show by genetic and pharmacological means that these drugs exert their effects through a different mechanism. Moreover, our results indicate that increases in cAMP levels and changes in PKA activity do not impact the circadian period and therefore are not part of underlying effects of methylxanthine. These results set the stage for future analyses dissecting the molecular mechanisms by which these drugs dramatically modify the circadian period.
Assuntos
Cafeína , Neurospora crassa , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/fisiologia , Ritmo Circadiano/efeitos dos fármacos , AMP Cíclico/metabolismo , Cafeína/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/antagonistas & inibidores , 1-Metil-3-Isobutilxantina , Proteínas Quinases/metabolismo , Transdução de SinaisRESUMO
Importin-α (Impα) is an adaptor protein that binds to cargo proteins (containing Nuclear Localization Sequences - NLSs), for their translocation to the nucleus. The specificities of the Impα/NLS interactions have been studied, since these features could be used as important tools to find potential NLSs in nuclear proteins or even for the development of targets to inhibit nuclear import or to design peptides for drug delivery. Few structural studies have compared different Impα variants from the same organism or Impα of different organisms. Previously, we investigated nuclear transport of transcription factors with Neurospora crassa Impα (NcImpα). Herein, NIT-2 and PAC-3 transcription factors NLSs were studied in complex with Mus musculus Impα (MmImpα). Calorimetric assays demonstrated that the PAC-3 NLS peptide interacts with both Impα proteins with approximately the same affinity. The NIT-2 NLS sequence binds with high affinity to the Impα major binding site from both organisms, but its binding to minor binding sites reveals interesting differences due to the presence of additional interactions of NIT-2-NLS with MmImpα. These findings, together with previous results with Impα from other organisms, indicate that the differential affinity of NLSs to minor binding sites may be also responsible for the selectivity of some cargo proteins recognition and transport.
Assuntos
Núcleo Celular/metabolismo , Camundongos/fisiologia , alfa Carioferinas/metabolismo , Aminoidrolases/genética , Aminoidrolases/metabolismo , Animais , Cristalização , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neurospora crassa/fisiologia , Sinais de Localização Nuclear/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Transporte Proteico , Transcrição Gênica , alfa Carioferinas/genéticaRESUMO
Agmatinase is known as a metalloenzyme which hydrolyzes agmatine to produce urea and putrescine, being crucial in the alternative pathway to produce polyamines. In this study, an agmatinase-like protein (AGM-1) (NCU 01348) in the filamentous fungus Neurospora crassa is reported. Purified AGM-1 from N. crassa displays enzymatic activity hydrolyzing agmatine; therefore, it can be considered as an agmatinase-like protein. However, its role in the alternative pathway to produce polyamines apparently is not its main function since only a slight reduction of polyamines concentration was detected in the Δagm-1 het strain. Moreover, the null mutant Δagm-1 (homokaryon strain) was unable to grow and the deficiency of agm-1 in the heterokaryon strain provoked a decrease in elongation rate, conidia and biomass production, despite of having de constitutive pathway via the ornithine decarboxylase (ODC). Additionally, mature hyphae of the Δagm-1 het strain presented unusual apical branching and a disorganized Spitzenkörper (Spk). Trying to reveal the role of AGM-1in N. crassa, the protein was tagged with GFP and interestingly the dynamics and intracellular localization of AGM-1 closely resembles the F-actin population. This finding was further examined in order to elucidate if AGM-1is in a close association with F-actin. Since polyamines, among them agmatine, have been reported to act as stabilizers of actin filaments, we evaluated in vitro G-actin polymerization in the presence of agmatine and the effect of purified AGM-1 from N. crassa on these polymerized actin filaments. It was found that polymerization of actin filaments increases in the presence of agmatine and the addition of purified AGM-1 from N. crassa depolymerizes these actin filaments. Also, it was determined that an intact substrate binding site of the enzyme is necessary for the localization pattern of the native AGM-1. These results suggest that in N. crassa AGM-1 has a close association with the F-actin population via its substrate agmatine, playing an essential role during cell development.
Assuntos
Agmatina/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/enzimologia , Ureo-Hidrolases/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas Fúngicas/genética , Hidrólise , Hifas/metabolismo , Neurospora crassa/genética , Neurospora crassa/fisiologia , Ureo-Hidrolases/genéticaRESUMO
Tic-tac, tic-tac, the sound of time is familiar to us, yet, it also silently shapes daily biological processes conferring 24-hour rhythms in, among others, cellular and systemic signaling, gene expression, and metabolism. Indeed, circadian clocks are molecular machines that permit temporal control of a variety of processes in individuals, with a close to 24-hour period, optimizing cellular dynamics in synchrony with daily environmental cycles. For over three decades, the molecular bases of these clocks have been extensively described in the filamentous fungus Neurospora crassa, yet, there have been few molecular studies in fungi other than Neurospora, despite evidence of rhythmic phenomena in many fungal species, including pathogenic ones. This chapter will revise the mechanisms underlying clock regulation in the model fungus N. crassa, as well as recent findings obtained in several fungi. In particular, this chapter will review the effect of circadian regulation of virulence and organismal interactions, focusing on the phytopathogen Botrytis cinerea, as well as several entomopathogenic fungi, including the behavior-manipulating species Ophiocordyceps kimflemingiae and Entomophthora muscae. Finally, this review will comment current efforts in the study of mammalian pathogenic fungi, while highlighting recent circadian lessons from parasites such as Trypanosoma and Plasmodium. The clock keeps on ticking, whether we can hear it or not.
Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Neurospora crassa/fisiologia , Neurospora crassa/patogenicidade , Animais , Humanos , VirulênciaRESUMO
Night follows day and as a consequence, organisms have evolved molecular machineries that allow them to anticipate and respond to the many changes that accompany these transitions. Circadian clocks are precise yet plastic pacemakers that allow the temporal organization of a plethora of biological process. Circadian clocks are widespread across the tree of life and while their exact molecular components differ among phyla, they tend to share common design principles. In this review, we discuss the circadian system of the filamentous fungus Neurospora crassa. Historically, this fungus has served a key role in the genetic and molecular dissection of circadian clocks, aiding in their detailed mechanistic understanding. Recent studies have provided new insights into the daily molecular dynamics that constitute the Neurospora circadian oscillator, some of which have questioned traditional paradigms describing timekeeping mechanisms in eukaryotes. In addition, recent reports support the idea of a dynamic network of transcription factors underlying the rhythmicity of thousands of genes in Neurospora, many of which oscillate only under specific conditions. Besides Neurospora, which harbors the best characterized circadian system among filamentous fungi, the recent characterization of the circadian system of the plant-pathogenic fungus Botrytis cinerea has provided additional insights into the physiological impact of the clock and potential additional functions of clock proteins in fungi. Finally, we speculate on the presence of FRQ or FRQ-like proteins in diverse fungal lineages.
Assuntos
Relógios Circadianos , Neurospora crassa/citologia , Neurospora crassa/fisiologia , Animais , Botrytis/fisiologia , Proteínas CLOCK/fisiologia , Ritmo CircadianoRESUMO
Chitin, one of the most important carbohydrates of the fungal cell wall, is synthesized by chitin synthases (CHS). Seven sequences encoding CHSs have been identified in the genome of Neurospora crassa. Previously, CHS-1, -3 and -6 were found at the Spitzenkörper(Spk) core and developing septa. We investigated the functional importance of each CHS in growth and development of N. crassa. The cellular distribution of each CHS tagged with fluorescent proteins and the impact of corresponding gene deletions on vegetative growth and sexual development were compared. CHS-2, -4, -5 and -7 were also found at the core of the Spk and in forming septa in vegetative hyphae. As the septum ring developed, CHS-2-GFP remained at the growing edge of the septum until it localized around the septal pore. In addition, all CHSs were located in cross-walls of conidiophores. A partial co-localization of CHS-1-m and CHS-5-GFP or CHS-2-GFP occurred in the Spk and septa. Analyses of deletion mutants suggested that CHS-6 has a role primarily in hyphal extension and ascospore formation, CHS-5 in aerial hyphae, conidia and ascospore formation, CHS-3 in perithecia development and CHS-7 in all of the aforementioned. We show that chs-7/csmB fulfills a sexual function and chs-6/chsG fulfills a vegetative growth function in N. crassa but not in Aspergillus nidulans, whereas vice versa chs-2/chsA fulfills a sexual function in A. nidulans but not in N. crassa. This suggests that different classes of CHSs can fulfill distinct developmental functions in various fungi. Immunoprecipitation followed by mass spectrometry of CHS-1-GFP, CHS-4-GFP and CHS-5-GFP identified distinct putative interacting proteins for each CHS. Collectively, our results suggest that there are distinct populations of chitosomes, each carrying specific CHSs, with particular roles during different developmental stages.
Assuntos
Quitina Sintase/fisiologia , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/genética , Aspergillus nidulans/genética , Vesículas Citoplasmáticas/fisiologia , Proteínas Fúngicas/genética , Genótipo , Proteínas de Fluorescência Verde/genética , Hifas/crescimento & desenvolvimento , Hifas/ultraestrutura , Imunoprecipitação , Neurospora crassa/fisiologia , Esporos Fúngicos/crescimento & desenvolvimento , Espectrometria de Massas em TandemRESUMO
All organisms are faced with environmental uncertainty. Bet-hedging theory expects unpredictable selection to result in the evolution of traits that maximize the geometric-mean fitness even though such traits appear to be detrimental over the shorter term. Despite the centrality of fitness measures to evolutionary analysis, no direct test of the geometric-mean fitness principle exists. Here, we directly distinguish between predictions of competing fitness maximization principles by testing Cohen's 1966 classic bet-hedging model using the fungus Neurospora crassa. The simple prediction is that propagule dormancy will evolve in proportion to the frequency of 'bad' years, whereas the prediction of the alternative arithmetic-mean principle is the evolution of zero dormancy as long as the expectation of a bad year is less than 0.5. Ascospore dormancy fraction in N. crassa was allowed to evolve under five experimental selection regimes that differed in the frequency of unpredictable 'bad years'. Results were consistent with bet-hedging theory: final dormancy fraction in 12 genetic lineages across 88 independently evolving samples was proportional to the frequency of bad years, and evolved both upwards and downwards as predicted from a range of starting dormancy fractions. These findings suggest that selection results in adaptation to variable rather than to expected environments.
Assuntos
Evolução Biológica , Meio Ambiente , Aptidão Genética , Neurospora crassa/fisiologia , Adaptação Fisiológica , África , Haiti , Neurospora crassa/genética , Estados UnidosRESUMO
Hyphae of the Ascomycota are tubular cells compartmentalized by perforated septa, whose central pore allows the flow of organelles and cytoplasm. While in plants and yeast septation leads to cell separation, in filamentous fungi the formation of crosswalls appears to have an architectural role, limits the extent of mechanical damage thus maintaining hyphal integrity, and also is of fundamental importance as part of cell differentiation. The increasing number of available fungal genome sequences, knockout mutants, versatile tools for protein tagging, and the continuous improvement of fluorescence microscopes have allowed scientists to analyze living cells and reveal the molecular and cellular basis of septation with unprecedented detail. This review summarizes the recent advances in septum ontogenesis in Neurospora crassa. A "septal actomyosin tangle" is the first indication of impending septation. It assembles prior to any visible evidence of plasma membrane inward growth, which occurs concomitantly with the formation and constriction of a contractile actomyosin ring and synthesis of the septum wall. One of the key questions in septum biogenesis is how the septation machinery is assembled to construct a centripetally growing crosswall. Most of the machinery utilized in apical cell wall growth can be expected at septation sites to ensure an organized arrival and supply of vesicles leading to the formation of a septum. Yet, the intrinsically different architecture of the septum may require a different organization and regulation of the wall-synthesizing machinery.
Assuntos
Parede Celular/metabolismo , Hifas/metabolismo , Neurospora crassa/metabolismo , Citoesqueleto de Actina/ultraestrutura , Transporte Biológico , Parede Celular/fisiologia , Parede Celular/ultraestrutura , Proteínas Fúngicas/metabolismo , Hifas/crescimento & desenvolvimento , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/fisiologiaRESUMO
Cell-cell fusion in sexually reproducing organisms is a mechanism to merge gamete genomes and, in multicellular organisms, it is a strategy to sculpt organs, such as muscle, bone, and placenta. Moreover, this mechanism has been implicated in pathological conditions, such as infection and cancer. Studies of genetic model organisms have uncovered a unifying principle: cell fusion is a genetically programmed process. This process can be divided in three stages: competence (cell induction and differentiation); commitment (cell determination, migration, and adhesion); and cell fusion (membrane merging and cytoplasmic mixing). Recent work has led to the discovery of fusogens, which are cell fusion proteins that are necessary and sufficient to fuse cell membranes. Two unrelated families of fusogens have been discovered, one in mouse placenta and one in Caenorhabditis elegans (syncytins and F proteins, respectively). Current research aims to identify new fusogens and determine the mechanisms by which they merge membranes.
Assuntos
Fusão Celular , Animais , Caenorhabditis elegans/fisiologia , Diferenciação Celular/fisiologia , Membrana Celular/fisiologia , Citoplasma/fisiologia , Feminino , Fertilização/genética , Fertilização/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/fisiologia , Humanos , Macrófagos/fisiologia , Fusão de Membrana/genética , Fusão de Membrana/fisiologia , Camundongos , Mioblastos/fisiologia , Neurospora crassa/fisiologia , Placenta/fisiologia , Plantas/metabolismo , Gravidez , Saccharomyces cerevisiae/fisiologiaRESUMO
A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk), whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture.
Assuntos
Polaridade Celular/fisiologia , Proteínas Fúngicas/análise , Proteínas dos Microfilamentos/análise , Microscopia/métodos , Neurospora crassa/ultraestrutura , Fusão Celular , Polaridade Celular/genética , Citocinese/genética , Citocinese/fisiologia , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/genética , Fungos/crescimento & desenvolvimento , Fungos/fisiologia , Fungos/ultraestrutura , Regulação Fúngica da Expressão Gênica , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Neurospora crassa/genética , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/fisiologia , Transporte Proteico , Regeneração/genética , Regeneração/fisiologia , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Esporos Fúngicos/fisiologia , Esporos Fúngicos/ultraestrutura , Distribuição TecidualRESUMO
The circadian rhythm of Neurospora crassa can be seen as a conidiation rhythm that produces concentric rings of bands (conidiating regions) alternating with interbands (non-conidiating regions) on the surface of an agar medium. To follow quantitatively this rhythm, densitometric analysis, gravimetric procedures, and video microscopy were employed. The circadian behavior of N. crassa is commonly monitored by cultivation in race tubes; in this work we report different growth kinetics during cultivation in conventional Petri dish cultures. Two different growth parameters were measured: total colony mass (true growth rate) and distance (colony radial expansion or hyphal elongation). Determinations of cellular mass revealed a dramatic circadian oscillation with a marked drop in growth rate during new interband formation followed by a sharp increase during the development of a new conidiation band. On the other hand, we found that the radial expansion of the colony previously reported to decrease periodically seemed unaffected by the circadian clock. Densitometric analysis showed no initial difference in the expanding margin of the colony, independent of whether that area was destined to be a band or an interband. The band areas increased rapidly in density for about 15 h whereas the interband areas maintained an equally rapid rate of increase for only 6h. The density of band areas kept increasing slowly for almost 40 h, along with an increase in the amount of conidia. Video microscopy showed the importance of cytoplasmic flow in colony development with continuous forward flow to support hyphal morphogenesis and reverse flow to support an extended period of conidiogenesis. Our results indicate that the circadian system of Neurospora can be expressed at the level of cellular mass formation, not just as the developmental conidiation rhythm.
Assuntos
Neurospora crassa/fisiologia , Ritmo Circadiano , Contagem de Colônia Microbiana , Hifas/crescimento & desenvolvimento , Microscopia de Vídeo , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/isolamento & purificação , Fatores de TempoRESUMO
Subtractive hybridization was used to isolate transcripts up-regulated in the nuc-2 mutant strain of Neurospora crassa grown under phosphate starvation. Following differential screening, 66 cDNA clones of the total enriched were screened in a second round by reverse Northern hybridization. The 17 cDNA candidates displaying visual positive differential expression were sequenced, and functional grouping identified putative proteins possibly involved in diverse cellular processes as, for example, protein synthesis, signal transduction mechanisms, and transport facilitation. Four of them, confirmed by both virtual and Northern blot analyses, revealed genes involved in the initiation of mRNA translation that are significantly up-regulated in the nuc-2 mutant strain, which may be relevant to a further understanding of the molecular events involved in the phosphorus sensing in N. crassa.
Assuntos
Repetição de Anquirina/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Mutação , Neurospora crassa/crescimento & desenvolvimento , Fosfatos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Neurospora crassa/fisiologia , Hibridização de Ácido Nucleico/métodos , Biossíntese de Proteínas , Transcrição Gênica , Regulação para CimaRESUMO
The influence of the cAMP-signalling pathway on the metabolism of trehalose in Neurospora crassa was investigated. The changes in intracellular trehalose concentration were measured in two mutants affected in components of the cAMP-signalling pathway: cr-1 (crisp-1), deficient in adenylyl cyclase activity, and mcb (microcyclic conidiation), deficient in the regulatory subunit of PKA. Rapid mobilisation of intracellular trehalose in the wild-type occurred, either at the onset of germination, or after a heat shock, and by carbon starvation. Mutant cr-1 failed to mobilise trehalose at germination, but behaved almost normally after a heat shock, or during carbon starvation. On the other hand, the levels of trehalose in mcb fell to values much lower than in the wild-type at germination, but accumulated trehalose normally during a heat shock. These results are consistent with the involvement of cAMP in the activation of the neutral trehalase at the onset of germination. However, the control of the enzyme under the other physiological conditions which also promote mobilisation of intracellular trehalose was apparently independent of cAMP-signalling.
Assuntos
Adenilil Ciclases/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , AMP Cíclico/metabolismo , Mutação , Neurospora crassa/metabolismo , Trealose/metabolismo , Adenilil Ciclases/metabolismo , Carbono/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Resposta ao Choque Térmico , Neurospora crassa/genética , Neurospora crassa/fisiologia , Transdução de SinaisRESUMO
We have cloned and sequenced a polyubiquitin gene from Neurospora crassa that is organized in a four repeat-tandem array. The first repeat contains a small intron and the last is fused to an extra glutamine codon. In Northern blots, two RNA species of 1.3 kb and 0.7 kb hybridize to the isolated clone. The larger ubiquitin (UBI) transcript accumulates after partial inhibition of protein synthesis with cycloheximide, and the smaller one preferentially accumulates in conidia after germination. Unexpectedly, constitutive expression of UBI transcripts in exponentially grown mycelia is not altered by heat-shock or exposure to arsenite.
Assuntos
Clonagem Molecular , Regulação da Expressão Gênica , Genes Fúngicos , Neurospora crassa/genética , Neurospora/genética , Ubiquitinas/genética , Sequência de Aminoácidos , Sequência de Bases , Códon , Cicloeximida/farmacologia , Sondas de DNA , Enzimas de Restrição do DNA , DNA Fúngico/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Íntrons , Dados de Sequência Molecular , Neurospora crassa/fisiologia , Hibridização de Ácido Nucleico , RNA Fúngico/genética , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
1. Pulse labeling with [35S]-methionine, one-dimensional SDS-polyacrylamide gel electrophoresis and fluorography were used to study the pattern of protein synthesis in Neurospora crassa mycelia undergoing sexual development. 2. Contact of sexually-competent mycelium with cells of the opposite mating type elicited a rapid and transient increase in the synthesis of two predominant proteins of 58 kDa and 40 kDa localized in the cytosol fraction. 3. Marked changes in the pattern of protein synthesis were also observed in the 12,000 g particulate fraction, predominantly mitochondrial, where the synthesis of a 34 kDa polypeptide was most prominent among others. 4. Poly(A)+ RNA extracted from mycelia 2 h after sexual stimulation supported the in vitro synthesis of the 58 kDa and 40 kDa major polypeptides synthesized in vivo. 5. No differences were observed in the pattern of protein synthesis of treated cultures and controls 24 h after the sexual stimulus.