Nickel induced cell impairments are negatively regulated by the Tor1 kinase in Schizosaccharomyces pombe.

In our study we investigated the effect of different nickel (NiSO4·6H2O) (Ni) concentrations on cell division, cellular morphology and ionome homeostasis of the eukaryotic model organism Schizosaccharomyces pombe. Target of rapamycin (TOR) protein kinase is one of the key regulators of cell growth under different environmental stresses. We analyzed the effect of Ni on cell strains lacking the Tor1 signaling pathway utilizing light-absorbance spectroscopy, visualization, microscopy and inductively coupled plasma optical emission spectroscopy. Interestingly, our findings revealed that Ni mediated cell growth alterations are noticeably lower in Tor1 deficient cells. Greater size of Tor1 depleted cells reached similar quantitative parameters to wild type cells upon incubation with 400 µM Ni. Differences of ion levels among the two tested yeast strains were detected even before Ni addition. Addition of high concentration (1 mM) of the heavy metal, representing acute contamination, caused considerable changes in the ionome of both strains. Strikingly, Tor1 deficient cells displayed largely reduced Ni content after treatment compared to wild type controls (644.1 ± 49 vs. 2096.8 ± 75 µg/g), suggesting its significant role in Ni trafficking. Together our results predict yet undefined role for the Tor1 signaling in metal uptake and/or metabolism.

The Role of Non-Catalytic Domains of Hrp3 in Nucleosome Remodeling.

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.

N-Terminus Does Not Govern Protein Turnover of Schizosaccharomyces pombe CENP-A.

Centromere integrity underlies an essential framework for precise chromosome segregation and epigenetic inheritance. Although centromeric DNA sequences vary among different organisms, all eukaryotic centromeres comprise a centromere-specific histone H3 variant, centromeric protein A (CENP-A), on which other centromeric proteins assemble into the kinetochore complex. This complex connects chromosomes to mitotic spindle microtubules to ensure accurate partitioning of the genome into daughter cells. Overexpression of CENP-A is associated with many cancers and is correlated with its mistargeting, forming extra-centromeric kinetochore structures. The mislocalization of CENP-A can be counteracted by proteolysis. The amino (N)-terminal domain (NTD) of CENP-A has been implicated in this regulation and shown to be dependent on the proline residues within this domain in Saccharomyces cerevisiae CENP-A, Cse4. We recently identified a proline-rich GRANT motif in the NTD of Schizosaccharomyces pombe CENP-A (SpCENP-A) that regulates the centromeric targeting of CENP-A via binding to the CENP-A chaperone Sim3. Here, we investigated whether the NTD is required to confer SpCENP-A turnover (i.e., counter stability) using various truncation mutants of SpCENP-A. We show that sequential truncation of the NTD did not improve the stability of the protein, indicating that the NTD of SpCENP-A does not drive turnover of the protein. Instead, we reproduced previous observations that heterochromatin integrity is important for SpCENP-A stability, and showed that this occurs in an NTD-independent manner. Cells bearing the null mutant of the histone H3 lysine 9 methyltransferase Clr4 (Δclr4), which have compromised constitutive heterochromatin integrity, showed reductions in the proportion of SpCENP-A in the chromatin-containing insoluble fraction of the cell extract, suggesting that heterochromatin may promote SpCENP-A chromatin incorporation. Thus, a disruption in heterochromatin may result in the delocalization of SpCENP-A from chromatin, thus exposing it to protein turnover. Taken together, we show that the NTD is not required to confer SpCENP-A protein turnover.

Branched unwinding mechanism of the Pif1 family of DNA helicases.

Members of the Pif1 family of helicases function in multiple pathways that involve DNA synthesis: DNA replication across G-quadruplexes; break-induced replication; and processing of long flaps during Okazaki fragment maturation. Furthermore, Pif1 increases strand-displacement DNA synthesis by DNA polymerase δ and allows DNA replication across arrays of proteins tightly bound to DNA. This is a surprising feat since DNA rewinding or annealing activities limit the amount of single-stranded DNA product that Pif1 can generate, leading to an apparently poorly processive helicase. In this work, using single-molecule Förster resonance energy transfer approaches, we show that 2 members of the Pif1 family of helicases, Pif1 from Saccharomyces cerevisiae and Pfh1 from Schizosaccharomyces pombe, unwind double-stranded DNA by a branched mechanism with 2 modes of activity. In the dominant mode, only short stretches of DNA can be processively and repetitively opened, with reclosure of the DNA occurring by mechanisms other than strand-switching. In the other less frequent mode, longer stretches of DNA are unwound via a path that is separate from the one leading to repetitive unwinding. Analysis of the kinetic partitioning between the 2 different modes suggests that the branching point in the mechanism is established by conformational selection, controlled by the interaction of the helicase with the 3' nontranslocating strand. The data suggest that the dominant and repetitive mode of DNA opening of the helicase can be used to allow efficient DNA replication, with DNA synthesis on the nontranslocating strand rectifying the DNA unwinding activity.

Functional and structural characterization of a novel catechol-O-methyltransferase from Schizosaccharomyces pombe.

Catechol-O-methyltransferase (COMT1 ) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to various catechol substrates. COMTs play vital roles in physiological processes in animals, plants, and fungi, as well as bacteria, and have essential application values in industry. spCOMT is a probable COMT from Schizosaccharomyces pombe. It has an extraordinary intracellular distribution different from other homologs and would thus be predicted to perform a distinct physiological function. In this report, recombinant spCOMT was purified and kinetically characterized for the first time. The enzymology assays indicate that spCOMT is a metal-dependent enzyme and belongs to class I OMTs. In addition, the crystal structures of apo-spCOMT and SAM-complexed spCOMT were also presented, revealing that spCOMT possesses a conserved SAM-binding site and Mg2+ pocket, but a distinct substrate pocket was not present in homologs. The mutagenesis ITC analysis revealed the SAM recognition characteristics of spCOMT. Based on all of the above findings, we speculated about the putative substrates' characteristics and the substrate recognition mechanisms of spCOMT. This work will help in elucidating the physiological functions of spCOMT in S. pombe. © 2018 IUBMB Life, 71(3):330-339, 2019.

Tetrad Dissection in Fission Yeast.

Tetrad dissection is a powerful tool in yeast genetics that allows the analysis of products of a single meiosis. With just a few tetrads, it is possible to determine linkage, identify unique phenotypes associated with double mutants, or assess specific meiotic defects. Strains are crossed on nitrogen-limiting medium for 3 days. With the help of a micromanipulator, ripe asci are isolated to spots 5 mm apart on a YES plate. Incubation at 36 °C for about 3-5 h is necessary for the ascus walls to break down. Once the spores are released, they are individually placed in a row containing four tetrad products, separated by 5 mm. The spores are then put in the appropriate temperature for the cross until colonies form, and phenotypes are assessed by replica plating or microscopic analysis.

Two three-strand intermediates are processed during Rad51-driven DNA strand exchange.

During homologous recombination, Rad51 forms a nucleoprotein filament with single-stranded DNA (ssDNA) that undergoes strand exchange with homologous double-stranded DNA (dsDNA). Here, we use real-time analysis to show that strand exchange by fission yeast Rad51 proceeds via two distinct three-strand intermediates, C1 and C2. Both intermediates contain Rad51, but whereas the donor duplex remains intact in C1, the ssDNA strand is intertwined with the complementary strand of the donor duplex in C2. Swi5-Sfr1, an evolutionarily conserved recombination activator, facilitates the C1-C2 transition and subsequent ssDNA release from C2 to complete strand exchange in an ATP-hydrolysis-dependent manner. In contrast, Ca2+, which activates the Rad51 filament by curbing ATP hydrolysis, facilitates the C1-C2 transition but does not promote strand exchange. These results reveal that Swi5-Sfr1 and Ca2+ have different activation modes in the late synaptic phase, despite their common function in stabilizing the presynaptic filament.

NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma-tubulin complex.

Mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ-tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha-helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N-terminus (residues 1-250) of GCP3 (γ-tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.

Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from Schizosaccharomyces pombe.

The tandem affinity purification (TAP) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. Proteins purified via the TAP tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. The original TAP tag contains a calmodulin-binding peptide and the IgG-binding domain from protein A separated by a tobacco etch virus (TEV) protease cleavage site. After capturing the Protein A epitope on an IgG resin, bound proteins are released by incubation with the TEV protease and then isolated on a calmodulin matrix in the presence of calcium; elution from this resin is achieved by chelating calcium with EGTA. However, because the robustness of the calmodulin-binding step in this procedure is highly variable, we replaced the calmodulin-binding peptide with three copies of the FLAG epitope, (3× FLAG)-TEV-Protein A, which can be isolated using an anti-FLAG resin. Elution from this matrix is achieved in the presence of an excess of a 3× FLAG peptide. In addition to allowing proteins to be released under mild conditions, elution by the 3× FLAG peptide adds an extra layer of specificity to the TAP procedure, because it liberates only FLAG-tagged proteins.

Fundamental mechanisms of telomerase action in yeasts and mammals: understanding telomeres and telomerase in cancer cells.

Aberrant activation of telomerase occurs in 85-90% of all cancers and underpins the ability of cancer cells to bypass their proliferative limit, rendering them immortal. The activity of telomerase is tightly controlled at multiple levels, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere, and finally activation and processivity. However, studies using cancer cell lines and other model systems have begun to reveal features of telomeres and telomerase that are unique to cancer. This review summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between normal cells and cancer cells, which may provide a foundation for telomere/telomerase targeted cancer treatments.

C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast Schizosaccharomyces pombe.

The mitotic arrest deficiency 2 (Mad2) protein is an essential component of the spindle assembly checkpoint that interacts with Cdc20/Slp1 and inhibit its ability to activate anaphase promoting complex/cyclosome (APC/C). In bladder cancer cell line the C-terminal residue of the mad2 gene has been found to be deleted. In this study we tried to understand the role of the C-terminal region of mad2 on the spindle checkpoint function. To envisage the role of C-terminal region of Mad2, we truncated 25 residues of Mad2 C-terminal region in fission yeast S.pombe and characterized its effect on spindle assembly checkpoint function. The cells containing C-terminal truncation of Mad2 exhibit sensitivity towards microtubule destabilizing agent suggesting perturbation of spindle assembly checkpoint. Further, the C-terminal truncation of Mad2 exhibit reduced viability in the nda3-KM311 mutant background at non-permissive temperature. Truncation in mad2 gene also affects its foci forming ability at unattached kinetochore suggesting that the mad2-∆CT mutant is unable to maintain spindle checkpoint activation. However, in response to the defective microtubule, only brief delay of mitotic progression was observed in Mad2 C-terminal truncation mutant. In addition we have shown that the deletion of two ß strands of Mad2 protein abolishes its ability to interact with APC activator protein Slp1/Cdc20. We purpose that the truncation of two ß strands (ß7 and ß8) of Mad2 destabilize the safety belt and affect the Cdc20-Mad2 interaction leading to defects in the spindle checkpoint activation.

The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines.

Iron homeostasis is tightly regulated since iron is an essential but toxic element in the cell. The GATA-type transcription factor Fep1 and its orthologs contribute to iron homeostasis in many fungi by repressing genes for iron uptake when intracellular iron is high. Even though the function and interaction partners of Fep1 have been elucidated extensively In Schizosaccharomyces pombe, the mechanism behind iron-sensing by Fep1 remains elusive. It has been reported that Fep1 interacts with Fe-S-containing monothiol glutaredoxin Grx4 and Grx4-Fra2 complex. In this study, we demonstrate that Fep1 also binds iron, in the form of Fe-S cluster. Spectroscopic and biochemical analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. The finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1.

Structure of the Dcp2-Dcp1 mRNA-decapping complex in the activated conformation.

The removal of the mRNA 5' cap (decapping) by Dcp2 shuts down translation and commits mRNA to full degradation. Dcp2 activity is enhanced by activator proteins such as Dcp1 and Edc1. However, owing to conformational flexibility, the active conformation of Dcp2 and the mechanism of decapping activation have remained unknown. Here, we report a 1.6-Å-resolution crystal structure of the Schizosaccharomyces pombe Dcp2-Dcp1 heterodimer in an unprecedented conformation that is tied together by an intrinsically disordered peptide from Edc1. In this ternary complex, an unforeseen rotation of the Dcp2 catalytic domain allows residues from both Dcp2 and Dcp1 to cooperate in RNA binding, thus explaining decapping activation by increased substrate affinity. The architecture of the Dcp2-Dcp1-Edc1 complex provides a rationale for the conservation of a sequence motif in Edc1 that is also present in unrelated decapping activators, thus indicating that the presently described mechanism of decapping activation is evolutionarily conserved.

Cloning, expression, purification and crystallization of Schizosaccharomyces pombe Set7, a putative histone methyltransferase.

Dysfunction of histone-modifying enzymes affects chromatin regulation and is involved in carcinogenesis, tumour progression and other diseases. Histone methyltransferases are a family of key histone-modifying enzymes, but their structures, functions and mechanisms are incompletely understood, thus constraining drug-design efforts. Here, preliminary steps towards structure-function studies of Schizosaccharomyces pombe Set7, a putative histone methyltransferase and the first yeast full-length SET-domain-containing protein to be studied using X-ray crystallography, are reported. The methods from cloning to X-ray diffraction and phasing are discussed and the results will aid in prospective studies of histone-modifying enzymes.

Data collection with a tailored X-ray beam size at 2.69 Å wavelength (4.6 keV): sulfur SAD phasing of Cdc23(Nterm).

The capability to reach wavelengths of up to 3.1 Å at the newly established EMBL P13 beamline at PETRA III, the new third-generation synchrotron at DESY in Hamburg, provides the opportunity to explore very long wavelengths to harness the sulfur anomalous signal for phase determination. Data collection at λ = 2.69 Å (4.6 keV) allowed the crystal structure determination by sulfur SAD phasing of Cdc23(Nterm), a subunit of the multimeric anaphase-promoting complex (APC/C). At this energy, Cdc23(Nterm) has an expected Bijvoet ratio〈|Fanom|〉/〈F〉of 2.2%, with 282 residues, including six cysteines and five methionine residues, and two molecules in the asymmetric unit (65.4 kDa; 12 Cys and ten Met residues). Selectively illuminating two separate portions of the same crystal with an X-ray beam of 50 µm in diameter allowed crystal twinning to be overcome. The crystals diffracted to 3.1 Å resolution, with unit-cell parameters a = b = 61.2, c = 151.5 Å, and belonged to space group P43. The refined structure to 3.1 Å resolution has an R factor of 18.7% and an Rfree of 25.9%. This paper reports the structure solution, related methods and a discussion of the instrumentation.

Copper(I) stabilization by cysteine/tryptophan motif in the extracellular domain of Ctr4.

Copper transporter Ctr4 of fission yeast has a quasi-palindromic sequence rich in cysteine and aromatic amino acid residues, CX4YWNWYX4C (where X represents any amino acid), in the N-terminal extracellular domain. A 24-mer peptide comprising this sequence is bound to Cu(I) through the cysteine thiolate coordination. Luminescence, UV absorption and resonance Raman spectra of the Cu(I)-peptide complex show that at least one of the two tryptophan side chains is located in close proximity to the thiolate-Cu(I) center and interacts with the Cu(I) ion via π-electrons of the indole ring. Although the thiolates and Cu(I) are oxidized to disulfide and Cu(II), respectively, only very slowly in air-saturated solutions, replacements of the tryptophan residues to phenylalanine significantly accelerate the oxidation reactions. The results obtained indicate that the interaction between Cu(I) and tryptophan via π-electrons plays a significant role in protecting the thiolate-Cu(I) center against the oxidation. The cysteine- and tryptophan-rich quasi-palindromic sequence may be a metal binding motif that stabilizes Cu(I) in the oxidizing extracellular environment.

A novel 3' splice site recognition by the two zinc fingers in the U2AF small subunit.

The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3' splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs). The two ZFs are positioned side by side on the ß sheet of the RRM domain. Further mutational analysis revealed that the ZFs bind cooperatively to the target RNA sequence, but the RRM domain acts simply as a scaffold to organize the ZFs and does not itself contact the RNA directly. This completely novel and unexpected mode of RNA-binding mechanism by the U2AF small subunit sheds light on splicing errors caused by mutations of this highly conserved protein.

Nile red fluorescence screening facilitating neutral lipid phenotype determination in budding yeast, Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe.

Investigation of yeast neutral lipid accumulation is important for biotechnology and also for modelling aberrant lipid metabolism in human disease. The Nile red (NR) method has been extensively utilised to determine lipid phenotypes of yeast cells via microscopic means. NR assays have been used to differentiate lipid accumulation and relative amounts of lipid in oleaginous species but have not been thoroughly validated for phenotype determination arising from genetic modification. A modified NR assay, first described by Sitepu et al. (J Microbiol Methods 91:321-328, 2012), was able to detect neutral lipid changes in Saccharomyces cerevisiae deletion mutants with sensitivity similar to more advanced methodology. We have also be able to, for the first time, successfully apply the NR assay to the well characterised fission yeast Schizosaccharomyces pombe, an increasingly important organism in biotechnology. The described NR fluorescence assay is suitable for increased throughput and rapid screening of genetically modified strains in both the biotechnology industry and for modelling ectopic lipid production for a variety of human diseases. This ultimately negates the need for labour intensive and time consuming lipid analyses of samples that may not yield a desirable lipid phenotype, whilst genetic modifications impacting significantly on the cellular lipid phenotype can be further promoted for more in depth analyses.

ATPase-dependent auto-phosphorylation of the open condensin hinge diminishes DNA binding.

Condensin, which contains two structural maintenance of chromosome (SMC) subunits and three regulatory non-SMC subunits, is essential for many chromosomal functions, including mitotic chromosome condensation and segregation. The ATPase domain of the SMC subunit comprises two termini connected by a long helical domain that is interrupted by a central hinge. The role of the ATPase domain has remained elusive. Here we report that the condensin SMC subunit of the fission yeast Schizosaccharomyces pombe is phosphorylated in a manner that requires the presence of the intact SMC ATPase Walker motif. Principal phosphorylation sites reside in the conserved, glycine-rich stretch at the hinge interface surrounded by the highly basic DNA-binding patch. Phosphorylation reduces affinity for DNA. Consistently, phosphomimetic mutants produce severe mitotic phenotypes. Structural evidence suggests that prior opening (though slight) of the hinge is necessary for phosphorylation, which is implicated in condensin's dissociation from and its progression along DNA.

Citrinin-induced fluidization of the plasma membrane of the fission yeast Schizosaccharomyces pombe.

Citrinin (CTN) is a toxic fungal metabolite that is a hazardous contaminant of foods and feeds. In the present study, its acute toxicity and effects on the plasma membrane of Schizosaccharomyces pombe were investigated. The minimum inhibitory concentration of CTN against the yeast cells proved to be 500 µM. Treatment with 0, 250, 500 or 1000 µM CTN for 60 min resulted in a 0%, 2%, 21% or 100% decrease, respectively, in the survival rate of the cell population. Treatment of cells with 0, 100, 500 or 1000 µM CTN for 20 min induced decrease in the phase-transition temperature of the 5-doxylstearic acid-labeled plasma membrane to 16.51, 16.04, 14.18 or 13.98°C, respectively as measured by electron paramagnetic resonance spectroscopy. This perturbation was accompanied by the efflux of essential K⁺ from the cells. The existence of an interaction between CTN and glutathione was detected for the first time by spectrofluorometry. Our observations may suggest a direct interaction of CTN with the free sulfhydryl groups of the integral proteins of the plasma membrane, leading to dose-dependent membrane fluidization. The change in fluidity disturbed the ionic homeostasis, contributing to the death of the cells, which is a novel aspect of CTN cytotoxicity.