Intracellular and Extracellular Cyclic Nucleotides in Wild-Type and White Collar Mutant Strains of Neurospora crassa

Abstract

Cyclic AMP and cyclic GMP were released into the growth medium of mycelia of Neurospora crassa wild-type strains St.L.74A and Em5297a and by white collar-i and white collar-2 mutant strains. After growth for 6 days at 18°C, there were 2.19 (St.L.74A), 5.83 (Em5297a), 1.38 (white collar-i), and 1.10 (white collar-2) nanomoles of cyclic AMP per gram dry weight of mycelia in the growth medium. These values corresponded to concentrations of cyclic AMP of between approximately 10 and 50 nanomolar. The corresponding values for extracellular cyclic GMP were typically less than 6% of the values for cyclic AMP. Following transfer to fresh medium, cyclic AMP efflux was demonstrated for each of the strains, and the amount of cyclic AMP exported into the fresh medium was greater at 25°C than 6°C. Intracellular cyclic AMP and cyclic GMP were also measured in each of the strains. The values for cyclic AMP were in the same range as those in the literature (approximately 0.5 to 1.5 nanomoles per gram dry weight of mycelia). However, the corresponding intracellular cyclic GMP values were less than 1% of the cyclic AMP values, i.e. more than 50 times lower than the value previously reported for the St.L.74A wild-type. Transfer of mycelia after 6 days at 18°C to fresh media and incubation for 2 hours at 25°C or 6°C did not consistently affect the intracellular level of cyclic AMP or cyclic GMP in the strains examined. We could detect no change in intracellular cyclic AMP when mycelia of the St.L.74A wild-type strain were irradiated with blue light for periods of up to 3.0 hours at 180 C, or in cyclic AMP and cyclic GMP for irradiation times of up to 1 minute at 6°C. We propose that the plasma membrane of Neurospora crassa is permeable to cyclic nucleotides, and the export of cyclic nucleotides into the growth medium may be a means of regulating intracellular levels. We conclude that three factors that affect carotenogenesis in Neurospora crassa (blue light, temperature, and the white collar mutations) have no appreciable effect on the total measurable intracellular cyclic nucleotides in this organism. There was no extracellular or intracellular cyclic AMP or cyclic GMP in the crisp-i mutant strain, which suggested either that adenylate cyclase (which is absent in cnisp-i) catalyzes the synthesis of both cyclic AMP and cyclic GMP or that the cnisp-i mutation somehow results in a deficiency of two enzymes (adenylate and guanylate cyclase).Cyclic AMP and cyclic GMP have important regulatory roles in the metabolism of animals, fungi, bacteria, lower plants, and possibly higher plants. In Francko [10]). In addition, in the slime mold Dictyostelium discoideum, extracellular cyclic AMP is involved in cellular interaction (for a review see Devreotes [9]).In Neurospora crassa the presence of cyclic GMP has been reported (25), and cyclic AMP may be involved in morphological development (24). The crisp-i morphological mutants can be restored to wild-type morphology by adding cyclic AMP to their growth medium (25,28). Work in this...

Signal Transduction In Fungi

“…dibutyryl cAMP (Jones and Bu'Lock, 1977;Cooper et al, 1985;Brunton and Gadd, 1989). Both cAMP and cGMP may be released from growing fungi into external medium with possible roles including a signal for limiting mycelial density, and/or the regulation of intracellular levels of these cyclic nucleotides (Shaw and Harding, 1987).…”

Involvement of protein kinase C in the response of Neurospora crassa to blue light

“…The possibility that cAMP participates in transduction of the blue-light signal has been explored (Shaw and Harding 1987;Kallies et al 1996), but different studies have led to con¯icting results, so that the involvement of cAMP remains questionable. Both the N. crassa adenylate cyclase (Kore-eda et al, 1991) and a member of the cAMP-dependent protein kinase family have been cloned (Yarden et al 1992), but no information suggesting a possible role of these enzymes in blue light signalling has been published.…”

Lessons from the Genome Sequence ofNeurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism

“…Two predicted cAMP phosphodiesterase genes are most similar to the highand low-affinity forms, respectively, found in S. cerevisiae (491,682). cAMP accumulates in the extracellular medium of wild-type Neurospora cultures (371,724), and it is has recently been shown that loss of the heterotrimeric G␣ gene, gna-1, blocks the ability of strains lacking a functional adenylyl cyclase or gna-3 G␣ gene to respond to cAMP supplementation (370,405). These data, coupled with the presence of GPCRs similar to slime mold cAMP receptors, suggest that cAMP or a related molecule may serve as an environmental signal and GPCR ligand in Neurospora.…”