WD40 Domain Divergence Is Important for Functional Differences between the Fission Yeast Tup11 and Tup12 Co-Repressor Proteins

We have previously demonstrated that subsets of Ssn6/Tup target genes have distinct requirements for the Schizosaccharomyces pombe homologs of the Tup1/Groucho/TLE co-repressor proteins, Tup11 and Tup12. The very high level of divergence in the histone interacting repression domains of the two proteins suggested that determinants distinguishing Tup11 and Tup12 might be located in this domain. Here we have combined phylogenetic and structural analysis as well as phenotypic characterization, under stress conditions that specifically require Tup12, to identify and characterize the domains involved in Tup12-specific action. The results indicate that divergence in the repression domain is not generally relevant for Tup12-specific function. Instead, we show that the more highly conserved C-terminal WD40 repeat domain of Tup12 is important for Tup12-specific function. Surface amino acid residues specific for the WD40 repeat domain of Tup12 proteins in different fission yeasts are clustered in blade 3 of the propeller-like structure that is characteristic of WD40 repeat domains. The Tup11 and Tup12 proteins in fission yeasts thus provide an excellent model system for studying the functional divergence of WD40 repeat domains.     

A comparison of three fission yeast mitochondrial genomes

The fission yeasts are members of the fungal order Schizosaccharomycetales, a candidate deep-diverging group within Ascomycota. Although a great deal of molecular information is available from Schizosaccharomyces pombe, a model eukaryote, very little is available from other members of this group. In order to better characterize mitochondrial genome evolution in this fungal lineage, the mitochondrial DNA (mtDNA) of two additional fission yeasts, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus var. japonicus, was sequenced. Whereas the mtDNA of S.pombe is only 19 431 bp, the mtDNA of S.octosporus is 44 227 bp, and that of S.japonicus var. japonicus is over 80 kb. The size variation of these mtDNAs is due largely to non-coding regions. The gene content in the latter two mtDNAs is almost identical to that of the completely sequenced S.pombe mtDNA, which encodes 25 tRNA species, the large and small mitochondrial ribosomal RNAs (rnl and rns), the RNA component of mitochondrial RNaseP (rnpB), mitochondrial small subunit ribosomal protein 3 (rps3), cytochrome oxidase subunits 1, 2 and 3 (cox1, cox2 and cox3) and ATP-synthase subunits 6, 8 and 9 (atp6, atp8 and atp9). However, trnI2(cau) (C modified to lysidine) is absent in the S.octosporus mtDNA, as are corresponding ATA codons in its protein-coding genes, and rps3 and rnpB are not found in the mtDNA of S.japonicus var. japonicus. The mtDNA of S.octosporus contains five double hairpin elements, the first report of these elements in an ascomycete. This study provides further evidence in favor of the mobility of these elements, and supports their role in mitochondrial genome rearrangement. The results of our phylogenetic analysis support the monophyly of the Schizosaccharomycetales, but question their grouping within the Archiascomycota.     

Analysis of the splicing machinery in fission yeast: a comparison with budding yeast and mammals

Based on genetic and bioinformatic analysis, 80 proteins from the newly sequenced Schizosaccharomyces pombe genome appear to be splicing factors. The fission yeast splicing factors were compared to those of Homo sapiens and Saccharomyces cerevisiae in order to determine the extent of conservation or divergence that has occurred over the billion years of evolution that separate these organisms. Our results indicate that many of the factors present in all three organisms have been well conserved throughout evolution. It is calculated that 38% of the fission yeast splicing factors are more similar to the human proteins than to the budding yeast proteins (>10% more similar or similar over a greater region). Many of the factors in this category are required for recognition of the 3′ splice site. Ten fission yeast splicing factors, including putative regulatory factors, have human homologs, but no apparent budding yeast homologs based on sequence data alone. Many of the budding yeast factors that are absent in fission yeast are associated with the U1 and U4/U6.U5 snRNP. Collectively the data presented in this survey indicate that of the two yeasts, S.pombe contains a splicing machinery more closely reflecting the archetype of a spliceosome.     

Purification, folding, and characterization of Rec12 (Spo11) meiotic recombinase of fission yeast

Meiotic recombination is initiated by controlled dsDNA breaks (DSBs). Rec12 (Spo11) protein of fission yeast is essential for the formation of meiotic DSBs in vivo, for meiotic recombination, and for segregation of chromosomes during meiosis I. Rec12 is orthologous to Top6A topoisomerase of Archaea and is likely the catalytic subunit of a meiotic recombinase that introduces recombinogenic DSBs. However, despite intensive effort, it has not been possible to produce Rec12 protein in a soluble form required to permit biochemical analyses of function. To obtain purified Rec12 protein for in vitro studies, a rec12(+) cDNA was generated, cloned into vector pET15b(+), and expressed in Escherichia coli. Rec12 protein was produced at moderate levels and it partitioned into insoluble fractions of whole-cell extracts. The protein was enriched based upon its differential solubility in two different denaturants and was further purified by column chromatography. A combinatorial, fractional, factorial approach was used to identify conditions under which Rec12 protein could be refolded. Four parameters were most important and, following optimization, soluble Rec12 protein was obtained. Gel filtration demonstrated that refolded Rec12 protein exists as a monomer in solution, suggesting that additional proteins may be required to assemble biologically-active Rec12 dimers, as inferred previously from genetic data [Cell Chromosome 1 (2002) 1]. The production of refolded Rec12 in a soluble form will allow for characterization in vitro of this key meiotic recombination enzyme.     

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.     

Functional homology among human and fission yeast Cdc14 phosphatases

Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis. In the present work, we show that human Cdc14 homologues (hCdc14A and hCdc14B) rescued flp1-deficient fission yeast strains, indicating functional homology. We also show that hCdc14A and B interacted in vivo with S. pombe Cdc25 and that hCdc14A dephosphorylated this mitotic inducer both in vitro and in vivo. Our results support a Cdc14 conserved inhibitory mechanism acting on S. pombe Cdc25 protein and suggest that human cells may regulate Cdc25 in a similar manner to inactivate Cdk1-mitotic cyclin complexes.     

Functional analysis of theDrosophila CDC2 Dm gene in fission yeast

Thecdc2 ⁺ gene product (p34^cdc2) is a protein kinase that regulates entry into mitosis in all eukaryotic cells. The role that p34^cdc2 plays in the cell cycle has been extensively investigated in a number of organisms, including the fission yeastSchizosaccharomyces pombe. To study the degree of functional conservation among evolutionarily distant p34^cdc2 proteins, we have constructed aS. pombe strain in which the yeastcdc2 ⁺ gene has been replaced by itsDrosophila homologue CDC2Dm (theCDC2Dm strain). ThisCDC2Dm S. pombe strain is viable, capable of mating and producing four viable meiotic products, indicating that the fly p34^CDC2Dm recognizes all the essentialS. pombe cdc2 ⁺ substrates, and that it is recognized by cyclin partners and other elements required for its activity. The p34^CDC2Dm protein yields a lethal phenotype in combination with the mutant B-type cyclin p56^cdc13-117, suggesting that thisS. pombe cyclin might interact less efficiently with theDrosophila protein than with its native p34^cdc2 counterpart. ThisCDC2Dm strain also responds to nutritional starvation and to incomplete DNA synthesis, indicating that proteins involved in these signal transduction pathways, interact properly with p34^CDC2Dm (and/or that p34^cdc2-independent pathways are used). TheCDC2Dm gene produces a ‘wee’ phenotype, and it is largely insensitive to the action of theS. pombe weel ⁺ mitotic inhibitor, suggesting thatDrosophila weel ⁺ homologue might not be functionally conserved. ThisCDC2Dm strain is hypersensitive to UV irradiation, to the same degree asweel-deficient mutants. A strain which co-expresses theDrosophila and yeastcdc2⁺ genes shows a dominantwee phenotype, but displays a wild-type sensitivity to UV irradiation, suggesting that p34^cdc2 triggers mitosis and influences the UV sensitivity by independent mechanisms.