Genetic defects in SAPK signalling, chromatin regulation, vesicle transport and CoA-related lipid metabolism are rescued by rapamycin in fission yeast.

Rapamycin inhibits TOR (target of rapamycin) kinase, and is being used clinically to treat various diseases ranging from cancers to fibrodysplasia ossificans progressiva. To understand rapamycin mechanisms of action more comprehensively, 1014 temperature-sensitive (ts) fission yeast (Schizosaccharomyces pombe) mutants were screened in order to isolate strains in which the ts phenotype was rescued by rapamycin. Rapamycin-rescued 45 strains, among which 12 genes responsible for temperature sensitivity were identified. These genes are involved in stress-activated protein kinase (SAPK) signalling, chromatin regulation, vesicle transport, and CoA- and mevalonate-related lipid metabolism. Subsequent metabolome analyses revealed that rapamycin upregulated stress-responsive metabolites, while it downregulated purine biosynthesis intermediates and nucleotide derivatives. Rapamycin alleviated abnormalities in cell growth and cell division caused by sty1 mutants (Δsty1) of SAPK. Notably, in Δsty1, rapamycin reduced greater than 75% of overproduced metabolites (greater than 2× WT), like purine biosynthesis intermediates and nucleotide derivatives, to WT levels. This suggests that these compounds may be the points at which the SAPK/TOR balance regulates continuous cell proliferation. Rapamycin might be therapeutically useful for specific defects of these gene functions.

CK2 phospho-independent assembly of the Tel2-associated stress-signaling complexes in Schizosaccharomyces pombe.

An evolutionarily conserved protein Tel2 regulates a variety of stress signals. In mammals, TEL2 associates with TTI1 and TTI2 to form the Triple T (TTT: TEL2-TTI1-TTI2) complex as well as with all the phosphatidylinositol 3-kinase-like kinases (PIKKs) and the R2TP (Ruvbl1-Ruvbl2-Tah1-Pih1 in budding yeast)/prefoldin-like complex that associates with HSP90. The phosphorylation of TEL2 by casein kinase 2 (CK2) enables direct binding of PIHD1 (mammalian Pih1) to TEL2 and is important for the stability and the functions of PIKKs. However, the regulatory mechanisms of Tel2 in fission yeast Schizosaccharomyces pombe remain largely unknown. Here, we report that S. pombe Tel2 is phosphorylated by CK2 at Ser490 and Thr493. Tel2 forms the TTT complex with Tti1 and Tti2 and also associates with PIKKs, Rvb2, and Hsp90 in vivo; however, the phosphorylation of Tel2 affects neither the stability of the Tel2-associated proteins nor their association with Tel2. Thus, Tel2 stably associates with its binding partners irrespective of its phosphorylation. Furthermore, the Tel2 phosphorylation by CK2 is not required for the various stress responses to which PIKKs are pivotal. Our results suggest that the Tel2-containing protein complexes are conserved among eukaryotes, but the molecular regulation of their formation has been altered during evolution.

The fission yeast spore is coated by a proteinaceous surface layer comprising mainly Isp3.

The spore is a dormant cell that is resistant to various environmental stresses. As compared with the vegetative cell wall, the spore wall has a more extensive structure that confers resistance on spores. In the fission yeast Schizosaccharomyces pombe, the polysaccharides glucan and chitosan are major components of the spore wall; however, the structure of the spore surface remains unknown. We identify the spore coat protein Isp3/Meu4. The isp3 disruptant is viable and executes meiotic nuclear divisions as efficiently as the wild type, but isp3∆ spores show decreased tolerance to heat, digestive enzymes, and ethanol. Electron microscopy shows that an electron-dense layer is formed at the outermost region of the wild-type spore wall. This layer is not observed in isp3∆ spores. Furthermore, Isp3 is abundantly detected in this layer by immunoelectron microscopy. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses.

Genetic control of cellular quiescence in S. pombe.

Transition from proliferation to quiescence brings about extensive changes in cellular behavior and structure. However, the genes that are crucial for establishing and/or maintaining quiescence are largely unknown. The fission yeast Schizosaccharomyces pombe is an excellent model in which to study this problem, because it becomes quiescent under nitrogen starvation. Here, we characterize 610 temperature-sensitive mutants, and identify 33 genes that are required for entry into and maintenance of quiescence. These genes cover a broad range of cellular functions in the cytoplasm, membrane and nucleus. They encode proteins for stress-responsive and cell-cycle kinase signaling pathways, for actin-bound and osmo-controlling endosome formation, for RNA transcription, splicing and ribosome biogenesis, for chromatin silencing, for biosynthesis of lipids and ATP, for cell-wall and membrane morphogenesis, and for protein trafficking and vesicle fusion. We specifically highlight Fcp1, a CTD phosphatase of RNA polymerase II, which differentially affects the transcription of genes that are involved in quiescence and proliferation. We propose that the transcriptional role of Fcp1 is central in differentiating quiescence from proliferation.

Rapamycin sensitivity of the Schizosaccharomyces pombe tor2 mutant and organization of two highly phosphorylated TOR complexes by specific and common subunits.

Nutrients are essential for cell growth and division. Screening of Schizosaccharomyces pombe temperature-sensitive strains led to the isolation of a nutrient-insensitive mutant, tor2-287. This mutant produces a nitrogen starvation-induced arrest phenotype in rich media, fails to recover from the arrest, and is hypersensitive to rapamycin. The L2048S substitution mutation in the catalytic domain in close proximity to the adenine base of ATP is unique as it is the sole known genetic cause of rapamycin hypersensitivity. Localization of Tor2 was speckled in the vegetative cytoplasm, and both speckled and membranous in the arrested cell cytoplasm. Using mass spectroscopic analysis, we identified six subunits (Tco89, Bit61, Toc1, Tel2, Tti1 and Cka1) that, in addition to the six previously identified subunits (Tor1, Tor2, Mip1/Raptor, Ste20/Rictor, Sin1/Avo1 and Wat1/Lst8), comprise the TOR complexes (TORCs). All of the subunits so far examined are multiply phosphorylated. Tel2 bound to Tti1 interacts with various phosphatidyl inositol kinase (PIK)-related kinases including Tra1, Tra2 and Rad3, as well as Tor1 and Tor2. Schizosaccharomyces pombe TORCs should thus be functionally redundant and might be broadly regulated through different subunits that are either common or specific to the two TORCs, or even common to various PIK-related kinases. Functional redundancy of the TORCs may explain the rapamycin hypersensitivity of tor2-287.

Two-step, extensive alterations in the transcriptome from G0 arrest to cell division in Schizosaccharomyces pombe.

Body cells in multicellular organisms are in the G0 state, in which cells are arrested and terminally differentiated. To understand how the G0 state is maintained, the genes that are specifically expressed or repressed in G0 must be identified, as they control G0. In the fission yeast Schizosaccharomyces pombe, haploid cells are completely arrested under nitrogen source starvation with high viability. We examined the global transcriptome of G0 cells and cells on the course to resume vegetative growth. Approximately 20% of the transcripts of approximately 5000 genes increased or decreased more than fourfold in the two-step transitions that occur prior to replication. Of the top 30 abundant transcripts in G0, 23 were replaced by ribosome- and translation-related transcripts in the dividing vegetative state. Eight identified clusters with distinct alteration patterns of approximately 2700 transcripts were annotated by Gene Ontology. Disruption of 53 genes indicated that nine of them were necessary to support the proper G0 state. These nine genes included two C2H2 zinc finger transcription factors, a cyclin-like protein implicated in phosphorylation of RNA polymerase II, two putative autophagy regulators, a G-protein activating factor, and two CBS domain proteins, possibly involved in AMP-activated kinase.