Fission yeast, Schizosaccharomyces pombe, is a natural inositol auxotroph. We show here that the amount of exogenous inositol added to the medium is critical for the control of its life cycle. Above growth-limiting concentrations inositol stimulates mating and sporulation in minimal medium. The effect of inositol is also observed on yeast-extract-medium plates. We selected a mutant, IM49, which mates and sporulates only poorly and show that it is defective in inositol transport. Its defect is in a gene (itr2) coding for a putative 12 membrane-spanning protein. The polypeptide contains the two sugar-transport motifs typical for hexose transporters and shows good homology to the two Saccharomyces cerevisiae inositol transporters. The itr2 gene is essential for cell growth and its mRNA level is repressed by glucose. Mutant IM49 is also complemented by a multicopy suppressor gene (itr1) which codes for a putative hexose transporter with unknown substrate specifity.
By screening gene libraries of Schizosaccharomyces pombe with a DNA fragment encoding part of the Saccharomyces cerevisiae S-adenosylmethionine synthetase (SAMS), we isolated the ®ssion yeast sam1 gene. Its sequence exhibits good homology to SAMSs of other organisms and reveals the motifs characteristic for SAMSs. SAMS activity and sam1 mRNA levels decrease when cells enter stationary phase. In haploid strains, gene sam1 is essential for growth; if weakly expressed, cells mate and sporulate at a reduced rate. Strains overexpressing sam1 exhibit methionine-sensitive growth. This methionine-induced growth inhibition is partially relieved by adenine. We assume that methionine reduces the level of one or several adenine nucleotides by a SAMS-mediated mechanism. Intracellular SAM levels increase drastically by exogenously added methionine. This increase predicts that mutants exhibiting methionine revertible phenotypes can be indicative for mutations in proteins exhibiting SAM-dependent functions. In agreement with this prediction, we show that mutant pmt2±5 has this phenotype and that gene pmt2 encodes a potential SAM-dependent enzyme.
CDC5 proteins are components of the pre-mRNA splicing complex and essential for cell cycle progression in yeast, plants and mammals. Human CDC5 is phosphorylated in a mitogen-dependent manner, and its association with the spliceosome is ATP-dependent. Examination of the amino acid sequence suggests that CDC5L may be phosphorylated at up to 28 potential consensus recognition sequences for known kinases, however, the identity of actual phosphorylation sites, their role in regulating CDC5L activity, and the kinases responsible for their phosphorylation have not previously been determined. Using two-dimensional phosphopeptide mapping and nanoelectrospray mass spectrometry, we now show that CDC5L is phosphorylated on at least nine sites in vivo. We demonstrate that while CDC5L is capable of forming homodimers in vitro and in vivo, neither homodimerization nor nuclear localization is dependent on phosphorylation at these sites. Using an in vitro splicing assay, we show that phosphorylation of CDC5L at threonines 411 and 438 within recognition sequences for CDKs are required for CDC5L-mediated pre-mRNA splicing. We also demonstrate that a specific inhibitor of CDK2, CVT-313, inhibits CDC5L phosphorylation in both in vitro kinase assays and in vivo radiolabeling experiments in cycling cells. These studies represent the first demonstration of a regulatory role for phosphorylation of CDC5L, and suggest that targeting these sites or the implicated kinases may provide novel strategies for treating disorders of unguarded cellular proliferation, such as cancer.
Genetic studies have shown that CDC5 proteins are essential for G2 progression and mitotic entry. CDC5 homologs in yeast and mammals are essential for pre-messenger ribonucleic acid (mRNA) processing. Other gene products also have been shown to play roles in both pre-mRNA splicing and cell cycle regulation, prompting the description of several models to explain the mechanism(s) linking these two processes. In this study, we demonstrate that the amino-terminus of human CDC5 directs nuclear import independent of consensus nuclear localization signals or phosphorylation, and that the carboxyl-terminus of human CDC5 preferentially associates with spliceosomal complexes in proximity of RNA transcription during interphase. hCDC5 colocalizes with Sm proteins in a cell cycle- and domain-dependent manner, and several proteins in the human CDC5-associated complex are identified that suggest potential roles for the complex in coupling pre-mRNA splicing to transcriptional activation and protein translation. These results indicate that human CDC5 may function in pre-mRNA processing and cell cycle progression through more than one mechanism.
It has previously been shown that the Schizosaccharomyces pombe mutant ksg1-358 has a mating and sporulation defect at 30 degrees C and that it is temperature sensitive for growth at 35 degrees C. However the molecular basis for these phenotypes remained largely unknown. In this study we show that ksg1-358 mutant cells lysed at the non-permissive temperature, which could be prevented by sorbitol. Overexpression of ksg1 using the nmt1-promoter showed slow growth and cells became swollen when incubated at 35 degrees C under low inositol conditions. Interestingly, in a two-hybrid assay we found that the ksg1-protein interacted with Pck1p, a protein implicated in regulating cell wall integrity in S. pombe. Genetic complementation assays showed that an overexpression of pck2, the homologue of pck1 involved in the regulation of cell wall synthesis, could partially rescue ksg1-358 phenotypes. We digested the ksg1-358 cell wall using beta-glucanase. We found that the ksg1-358 mutant was more resistant to cell lysis at 30 degrees C than the wildtype strain h972, which was similar to a pck1-deletion strain. A ksg1-overexpressing strain was hypersensitive towards beta-glucanase treatment similar to a pck2-deletion strain. The pck1-deletion partially rescued beta-glucanase hypersensitivity of the ksg1-overexpressing strain but the pck2-deletion increased it. The ksg1-358 mutation increased beta-glucanase resistance of a pck1-overexpressing strain but it had no effect on a pck2-overexpressing strain. Our results provide evidence that ksg1 is a novel regulator of cell wall integrity in the fission yeast Schizosaccharomyces pombe. They further suggest that Ksg1p acts in a pathway with Pck1p, possibly upstream and through direct interaction.
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