Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast

Summary Schizosaccharomyces pombe initiates sexual development in response to nutritional starvation. The level of cAMP inS. pombe cells changed during the transition from exponential growth to stationary phase. It also changed in response to a shift from nitrogen-rich medium to nitrogen-free medium. A decrease of approximately 50% was observed in either case, suggesting thatS. pombe cells contain less cAMP when they initiate sexual development.S. pombe cells that expressed the catalytic domain ofSaccharomyces cerevisiae adenylyl cyclase from theS. pombe adh1 promoter contained 5 times as much cAMP as the wild type and could not initiate mating and meiosis. These observations, together with previous findings that exogenously added cAMP inhibits mating and meiosis and that cells with little cAMP are highly derepressed for sexual development, strongly suggest that cAMP functions as a key regulator of sexual development inS. pombe. Thepde1 gene, which encodes a protein homologous toS. cerevisiae cAMP phosphodiesterase I, was isolated as a multicopy suppressor of the sterility caused by a high cAMP level. Disruption ofpde1 madeS. pombe cells partially sterile and meiosis-deficient, indicating that this cAMP phosphodiesterase plays an important role in balancing the cAMP level in vivo.     

Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor

The zygomycete Mucor circinelloides is an opportunistic fungal pathogen that commonly infects patients with malignancies, diabetes mellitus, and solid organ transplants. Despite the widespread use of antifungal therapy in the management of zygomycosis, the incidence of infections continues to rise among immunocompromised individuals. In this study, we established that the target and mechanism of antifungal action of the immunosuppressant rapamycin in M. circinelloides are mediated via conserved complexes with FKBP12 and a Tor homolog. We found that spontaneous mutations that disrupted conserved residues in FKBP12 conferred rapamycin and FK506 resistance. Disruption of the FKBP12-encoding gene, fkbA, also conferred rapamycin and FK506 resistance. Expression of M. circinelloides FKBP12 (McFKBP12) complemented a Saccharomyces cerevisiae mutant strain lacking FKBP12 to restore rapamycin sensitivity. Expression of the McTor FKBP12-rapamycin binding (FRB) domain conferred rapamycin resistance in S. cerevisiae, and McFKBP12 interacted in a rapamycin-dependent fashion with the McTor FRB domain in a yeast two-hybrid assay, validating McFKBP12 and McTor as conserved targets of rapamycin. We showed that in vitro, rapamycin exhibited potent growth inhibitory activity against M. circinelloides. In a Galleria mellonella model of systemic mucormycosis, rapamycin improved survival by 50%, suggesting that rapamycin and nonimmunosuppressive analogs have the potential to be developed as novel antifungal therapies for treatment of patients with mucormycosis.     

Establishment of a cellular axis in fission yeast

Recent studies in fission yeast Schizosaccharomyces pombe reveal how cells establish a cellular axis that specifies domains as the functional 'ends' and 'middle' of the cell. During interphase, dynamic microtubules position the nucleus at the middle of the cell and orientate microtubule 'plus' ends towards the ends of the cell. At the cell ends, the microtubule plus ends might establish a zone of polarized cell growth and actin assembly by depositing factors such as Tea1p. At the cell middle, the nucleus might specify the position of the actin contractile ring and the future cell division site by positioning cytokinesis factors such as Mid1p.     

Rapamycin specifically interferes with the developmental response of fission yeast to starvation.

Rapamycin is a microbial macrolide which belongs to a family of immunosuppressive drugs that suppress the immune system by blocking stages of signal transduction in T lymphocytes. In Saccharomyces cerevisiae cells, as in T lymphocytes, rapamycin inhibits growth and cells become arrested at the G1 stage of the cell cycle. Rapamycin is also an effective antifungal agent, affecting the growth of yeast and filamentous fungi. Unexpectedly, we observed that rapamycin has no apparent effect on the vegetative growth of Schizosaccharomyces pombe. Instead, the drug becomes effective only when cells experience starvation. Under such conditions, homothallic wild-type cells will normally mate and undergo sporulation. In the presence of rapamycin, this sexual development process is strongly inhibited and cells adopt an alternative physiological option and enter stationary phase. Rapamycin strongly inhibits sexual development of haploid cells prior to the stage of sexual conjugation. In contrast, the drug has only a slight inhibitory effect on the sporulation of diploid cells. A genetic approach was applied to identify the signal transduction pathway that is inhibited by rapamycin. The results indicate that either rapamycin did not suppress the derepression of sexual development of strains in which adenylate cyclase was deleted or the cyclic AMP-dependent protein kinase encoded by pka1 was mutated. Nor did rapamycin inhibit the unscheduled meiosis observed in pat1-114 mutants. Overexpression of ras1+, an essential gene for sexual development, did not rescue the sterility of rapamycin-treated cells. However, expression of the activated allele, ras1Val17, antagonized the effect of rapamycin and restored the ability of the cells to respond to mating signals in the presence of the drug. We discuss possible mechanisms for the inhibitory effect of rapamycin on sexual development in S. pombe.     

Loss of the TOR kinase Tor2 mimics nitrogen starvation and activates the sexual development pathway in fission yeast

Fission yeast has two TOR (target of rapamycin) kinases, namely Tor1 and Tor2. Tor1 is required for survival under stressed conditions, proper G(1) arrest, and sexual development. In contrast, Tor2 is essential for growth. To analyze the functions of Tor2, we constructed two temperature-sensitive tor2 mutants. Interestingly, at the restrictive temperature, these mutants mimicked nitrogen starvation by arresting the cell cycle in G(1) phase and initiating sexual development. Microarray analysis indicated that expression of nitrogen starvation-responsive genes was induced extensively when Tor2 function was suppressed, suggesting that Tor2 normally mediates a signal from the nitrogen source. As with mammalian and budding yeast TOR, we find that fission yeast TOR also forms multiprotein complexes analogous to TORC1 and TORC2. The raptor homologue, Mip1, likely forms a complex predominantly with Tor2, producing TORC1. The rictor/Avo3 homologue, Ste20, and the Avo1 homologue, Sin1, appear to form TORC2 mainly with Tor1 but may also bind Tor2. The Lst8 homologue, Wat1, binds to both Tor1 and Tor2. Our analysis shows, with respect to promotion of G(1) arrest and sexual development, that the loss of Tor1 (TORC2) and the loss of Tor2 (TORC1) exhibit opposite effects. This highlights an intriguing functional relationship among TOR kinase complexes in the fission yeast Schizosaccharomyces pombe.     

An STE12 homolog from the asexual, dimorphic fungus Penicillium marneffei complements the defect in sexual development of an Aspergillus nidulans steA mutant

Penicillium marneffei is an opportunistic fungal pathogen of humans and the only dimorphic species identified in its genus. At 25 degrees P. marneffei exhibits true filamentous growth, while at 37 degrees P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. Members of the STE12 family of regulators are involved in controlling mating and yeast-hyphal transitions in a number of fungi. We have cloned a homolog of the S. cerevisiae STE12 gene from P. marneffei, stlA, which is highly conserved. The stlA gene, along with the A. nidulans steA and Cryptococcus neoformans STE12alpha genes, form a distinct subclass of STE12 homologs that have a C2H2 zinc-finger motif in addition to the homeobox domain that defines STE12 genes. To examine the function of stlA in P. marneffei, we isolated a number of mutants in the P. marneffei-type strain and, in combination with selectable markers, developed a highly efficient DNA-mediated transformation procedure and gene deletion strategy. Deletion of the stlA gene had no detectable effect on vegetative growth, asexual development, or dimorphic switching in P. marneffei. Despite the lack of a detectable function, the P. marneffei stlA gene complemented the sexual defect of an A. nidulans steA mutant. In addition, substitution rate estimates indicate that there is a significant bias against nonsynonymous substitutions. These data suggest that P. marneffei may have a previously unidentified cryptic sexual cycle.     

Inositol is specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a natural auxotroph for inositol and fails to grow in the complete absence of it. It was previously reported that a small concentration of inositol in the culture medium supports vegetative growth, but not mating and sporulation, and a tenfold of that concentration also supports mating and sporulation. The purpose of the present work was to investigate whether a moderate inositol starvation specifically affected events of the sexual program of development. A homothallic culture grown to the stationary phase in medium with a small inositol concentration was sterile but cells in the stationary phase of growth synchronously entered and completed the sexual cycle when inositol was added, without need of previous cell divisions. This suggests the involvement of inositol in a mechanism (or mechanisms) of the sexual program. The events of the program that were affected by inositol starvation were investigated. Commitment to mating and production of pheromone M were shown not to be inositol-dependent. A diploid strain homozygous at the mating-type locus and carrying a pat1-114 temperature-sensitive mutation in homozygous configuration sporulated under inositol starvation at the restrictive temperature; therefore starvation did not directly affect meiosis or sporulation. In contrast, production of pheromone P and the response of cells to pheromones were found to be inositol-dependent. The possibility that inositol or one of its derivative compounds is involved in pheromone P secretion and in pheromone signal reception is discussed.     

Hmo1p, a high mobility group 1/2 homolog, genetically and physically interacts with the yeast FKBP12 prolyl isomerase

The immunosuppressive drugs FK506 and rapamycin bind to the cellular protein FKBP12, and the resulting FKBP12-drug complexes inhibit signal transduction. FKBP12 is a ubiquitous, highly conserved, abundant enzyme that catalyzes a rate-limiting step in protein folding: peptidyl-prolyl cis-trans isomerization. However, FKBP12 is dispensible for viability in both yeast and mice, and therefore does not play an essential role in protein folding. The functions of FKBP12 may involve interactions with a number of partner proteins, and a few proteins that interact with FKBP12 in the absence of FK506 or rapamycin have been identified, including the ryanodine receptor, aspartokinase, and the type II TGF-beta receptor; however, none of these are conserved from yeast to humans. To identify other targets and functions of FKBP12, we have screened for mutations that are synthetically lethal with an FKBP12 mutation in yeast. We find that mutations in HMO1, which encodes a high mobility group 1/2 homolog, are synthetically lethal with mutations in the yeast FPR1 gene encoding FKBP12. Deltahmo1 and Deltafpr1 mutants share two phenotypes: an increased rate of plasmid loss and slow growth. In addition, Hmo1p and FKBP12 physically interact in FKBP12 affinity chromatography experiments, and two-hybrid experiments suggest that FKBP12 regulates Hmo1p-Hmo1p or Hmo1p-DNA interactions. Because HMG1/2 proteins are conserved from yeast to humans, our findings suggest that FKBP12-HMG1/2 interactions could represent the first conserved function of FKBP12 other than mediating FK506 and rapamycin actions.     

Budding yeast Cdc6p induces re-replication in fission yeast by inhibition of SCFPop-mediated proteolysis

In fission yeast, overexpression of the replication initiator protein Cdc18p induces re-replication, a phenotype characterized by continuous DNA synthesis in the absence of cell division. In contrast, overexpression of Cdc6p, the budding yeast homolog of Cdc18p, does not cause re-replication in S. cerevisiae. However, we have found that Cdc6p has the ability to induce re-replication in fission yeast. Cdc6p cannot functionally replace Cdc18p, but instead interferes with the proteolysis of both Cdc18p and Rum1p, the inhibitor of the protein kinase Cdc2p. This activity of Cdc6p is entirely contained within a short N-terminal peptide, which forms a tight complex with Cdc2p and the F-box/WD-repeat protein Sud1p/Pop2p, a component of the SCF^Pop ubiquitin ligase in fission yeast. These interactions are mediated by two distinct regions within the N-terminal region of Cdc6p and depend on the integrity of its Cdc2p phosphorylation sites. The data suggest that disruption of re-replication control by overexpression of Cdc6p in fission yeast is a consequence of sequestration of Cdc2p and Pop2p, two factors involved in the negative regulation of Rum1p, Cdc18p and potentially other replication proteins.     

A homolog of mammalian antizyme is present in fission yeast Schizosaccharomyces pombe but not detected in budding yeast Saccharomyces cerevisiae

MOTIVATION: The antizymes (AZ) are proteins that regulate cellular polyamine pools in metazoa. To search for remote homologs in single-celled eukaryotes, we used computer software based on hidden Markov models. The most divergent homolog detected was that of the fission yeast Schizosaccharomyces pombe. Sequence identities between S.POMBE: AZ and known AZs are as low as 18-22% in the most conserved C-terminal regions. The authenticity of the S.POMBE: AZ is validated by the presence of a conserved nucleotide sequence that, in metazoa, promotes a +1 programmed ribosomal frameshift required for AZ expression. However, no homolog was detected in the completed genome of the budding yeast Saccharomyces cerevisiae. Procedural details and supplementary information can be found at http://itsa.ucsf.edu/ approximately czhu/AZ.     

Regulation and targeting of the fission yeast formin cdc12p in cytokinesis

Formins are conserved actin nucleators which promote the assembly of actin filaments for the formation of diverse actin structures. In fission yeast Schizosaccharomyces pombe, the formin cdc12p is required specifically in assembly of the actin-based contractile ring during cytokinesis. Here, using a mutational analysis of cdc12p, we identify regions of cdc12p responsible for ring assembly and localization. Profilin-binding residues of the FH1 domain regulate actin assembly and processive barbed-end capping by the FH2 domain. Studies using photobleaching (FRAP) and sensitivity to latrunculin A treatment show that profilin binding modulates the rapid dynamics of actin and cdc12p within the ring in vivo. Visualized by functional GFP-fusion constructs expressed from the endogenous promoter, cdc12p appears in a small number of cytoplasmic motile spot structures that deliver the formin to the ring assembly site, without detectable formation of an intermediate band of "nodes." The FH3/DID region directs interphase spot localization, while an N-terminal region and the FH1-FH2 domains of cdc12p can target its localization to the ring. Mutations in putative DID and DAD regions do not alter regulation, suggesting that cdc12p is not regulated by a canonical autoinhibition mechanism. Our findings provide insights into the regulation of formin activity and the mechanisms of contractile ring dynamics and assembly.     

Its8, a fission yeast homolog of Mcd4 and Pig-n, is involved in GPI anchor synthesis and shares an essential function with calcineurin in cytokinesis

In fission yeast, calcineurin is required for cytokinesis and ion homeostasis; however, most of its physiological roles remain obscure. To identify genes that share an essential function with calcineurin, we screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and high temperature and isolated the mutant its8-1. its8(+) encodes a homolog of the budding yeast MCD4 and human Pig-n that are involved in glycosylphosphatidylinositol (GPI) anchor synthesis. Consistently, reduced inositol labeling of proteins suggested impaired GPI anchor synthesis in its8-1 mutants. The temperature upshift induced a further decrease in inositol labeling and caused dramatic increases in the frequency of septation in its8-1 mutants. BE49385A, an inhibitor of MCD4 and Pig-n, also increased the septation index of the wild-type cell. Osmotic stabilization suppressed these morphological defects, indicating that cell wall weakness caused by impaired GPI anchor synthesis resulted in abnormal cytokinesis. Furthermore, calcineurin-deleted cells exhibited hypersensitivity to BE49385A, and FK506 exacerbated the cytokinesis defects of the its8-1 mutant. Thus, calcineurin and Its8 may share an essential function in cytokinesis and cell viability through the regulation of cell wall integrity.     

Involvement of ras in sexual differentiation but not in growth control in fission yeast

The function of the ras⁺ gene of Schizosaccharomyces pombe has been studied by constructing null and activated alleles of this gene. An activated allele (^rasVal ₁₂) inhibits conjugation but has no effect on cell growth, entry into stationary phase or sporulation. The phenotype of ^rasVal ₁₂ is distinct from that caused by elevating the intracellular level of cAMP. This supports the hypothesis that ras of fission yeast does not modulate adenylate cyclase in a manner analogous to S. cerevisiae RAS. Introduction of a human ras sequence into fission yeast cells containing a non-functional null allele of ras restored the sexual differentiation process thus indicating that the human sequence can complement S. pombe ras. Our data suggest that although ras genes are highly conserved across a considerable evolutionary divide, the cellular function of the ras gene product varies in different organisms.