Fission Yeast Protein Research Articles

The involvement of  theSchizosaccharomyces pombe sep9/spt8+gene in the regulation of septum cleavage

Schizosaccharomyces cells divide by medial septation, followed by enzymatic degradation of parts of the septum (septum cleavage) to allow the sister cells to separate from each other. In a previous study we found that the cell separation mutant sep9-307 was defective in a gene that encodes a protein highly similar in sequence to the Saccharomyces cerevisiae protein Spt8, a subunit of the SAGA complex. Here, we show that the sep9-307 mutation causes a frameshift in translation. The deletion of sep9(+) is not lethal but abolishes normal septum cleavage by reducing the activity of ace2(+), a gene coding for a transcription factor of numerous genes producing proteins for septum cleavage. Indirect evidence indicates that Sep9 might also act directly in the transcription of certain target genes (e.g. eng1(+)) of this regulator. sep9-307 is synthetically lethal with mutations in the cell separation genes sep11/med18(+) and sep15/med8(+), which encode subunits of the general transcription factor mediator. Heterologous expression of SPT8 and the putative Schizosaccharomyces japonicus sep9(+) orthologue cannot substitute for sep9(+). Both Spt8 and the fission yeast proteins have highly acidic (74-76 amino-acid long) N-terminal regions with no sequence conservation.

Architecture of the Smc5/6 Complex of Saccharomyces cerevisiae Reveals a Unique Interaction between the Nse5-6 Subcomplex and the Hinge Regions of Smc5 and Smc6

The evolutionarily conserved structural maintenance of chromosome (SMC) proteins forms the core structures of three multisubunit complexes as follows: cohesin, condensin, and the Smc5/6 complex. These complexes play crucial roles in different aspects of chromosomal organization, duplication, and segregation. Although the architectures of cohesin and condensin are better understood, that of the more recently identified Smc5/6 complex remains to be elucidated. We have previously shown that the Smc5/6 complex of Saccharomyces cerevisiae contains Smc5, Smc6, and six non-SMC elements (Nse1-6). In this study, we investigated the architecture of the budding yeast Smc5/6 complex employing the yeast two-hybrid assay as well as in vitro biochemical approaches using purified recombinant proteins. These analyses revealed that Smc5 and Smc6 associate with each other at their hinge regions and constitute the backbone of the complex, whereas the Nse1-6 subunits form three distinct subcomplexes/entities that interact with different regions of Smc5 and Smc6. The Nse1, -3, and -4 subunits form a stable subcomplex that binds to the head and the adjacent coiled-coil region of Smc5. Nse2 binds to the middle of the coiled-coil region of Smc5. Nse5 and Nse6 interact with each other and, as a heterodimer, bind to the hinge regions of Smc5 and Smc6. These findings provide new insights into the structures of the Smc5/6 complex and lay the foundation for further investigations into the mechanism of its functions.

Towards Mapping Domain Boundaries of Proteins

GFP fluorescence from bacterial colonies expressing a polypeptide upstream of GFP depends on the solubility of the polypeptide. Inspired by this idea, we developed a method to test for the folding of any polypeptide longer than about 66 amino acids. This method bypasses the need for purification of the polypeptide to test for folding. We employed this technique to identify the independently folded domains of Mid1, a fission yeast protein of 920 amino acids that participate in cytokinesis. A random fragment library of Mid1 cDNA was generated using tagged random primer PCR (tPCR). The fragment library was cloned upstream of a monomeric enhanced GFP (mEGFP) and expressed in E.coli. The GFP fluorescence of the bacterial colonies expressing the fusion constructs were compared to a threshold level of fluorescence to test if the Mid1 fragment fused to mEGFP is folded. Colonies expressing folded fragments of Mid1 are brighter than control colonies or colonies expressing an unfolded protein fragment fused upstream of mEGFP. Approximately 27000 colonies were screened to identify five soluble folded domains and one insoluble region, accounting for the entire length of Mid1 protein. This large scale approach will be useful in rapidly mapping domain boundaries of proteins in absence of any prior knowledge about the domain organization of the proteins.

Rapid regulation of protein activity in fission yeast

BackgroundThe fission yeast Schizosaccharomyces pombe is widely-used as a model organism for the study of a broad range of eukaryotic cellular processes such as cell cycle, genome stability and cell morphology. Despite the availability of extensive set of genetic, molecular biological, biochemical and cell biological tools for analysis of protein function in fission yeast, studies are often hampered by the lack of an effective method allowing for the rapid regulation of protein level or protein activity.ResultsIn order to be able to regulate protein function, we have made use of a previous finding that the hormone binding domain of steroid receptors can be used as a regulatory cassette to subject the activity of heterologous proteins to hormonal regulation. The approach is based on fusing the protein of interest to the hormone binding domain (HBD) of the estrogen receptor (ER). The HBD tag will attract the Hsp90 complex, which can render the fusion protein inactive. Upon addition of estradiol the protein is quickly released from the Hsp90 complex and thereby activated. We have tagged and characterised the induction of activity of four different HBD-tagged proteins. Here we show that the tag provided the means to effectively regulate the activity of two of these proteins.ConclusionThe estradiol-regulatable hormone binding domain provides a means to regulate the function of some, though not all, fission yeast proteins. This system may result in very quick and reversible activation of the protein of interest. Therefore it will be a powerful tool and it will open experimental approaches in fission yeast that have previously not been possible. Since fission yeast is a widely-used model organism, this will be valuable in many areas of research.

C. elegans Telomeres Contain G-Strand and C-Strand Overhangs that Are Bound by Distinct Proteins

Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in C. elegans, chromosome termini possess 3' G-strand overhangs as well as 5' C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that C. elegans telomere homeostasis relies on a novel mechanism that involves 5' and 3' single-stranded termini.

The 3′ Ends of Mature Transcripts Are Generated by a Processosome Complex in Fission Yeast Mitochondria

In this article, we report on the genetic analysis of the Schizosaccharomyces pombe open reading frames SPCC1322.01 and SPAC637.11, respectively, which encode proteins that are similar to the exoribonuclease Dss1p and the RNA helicase Suv3p, respectively, forming the mitochondrial degradosome of Saccharomyces cerevisiae. While the helicase Suv3p is exchangeable between S. cerevisiae and S. pombe, the functions of Dss1p and the putative fission yeast RNase protein are specific for each species. Unlike S. cerevisiae mutants lacking a functional degradosome, the major defect of fission yeast knock-out strains is their inability to perform downstream processing of transcripts. In addition, the lack of pah1 results in instability of mitochondrial RNA ends. Overexpression of par1 and pah1 has no significant effect on the steady-state levels of mitochondrial RNAs. The Pet127p-stimulated RNA degradation activity is independent of Par1p/Pah1p in fission yeast mitochondria. The results presented herein indicate that both fission yeast proteins play only a minor role (if at all) in mitochondrial RNA degradation. We assume that the RNA-degrading function was taken over by other enzymes in fission yeast mitochondria, while the former degradosome proteins were recruited to new cellular pathways, for example, RNA processing in fission yeast (as discussed in this article) or mitochondrial DNA replication, apoptosis, or chromatin maintenance in eukaryotes, during evolution.

Reconstitution of a microtubule plus-end tracking system in vitro

The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

Convergent Domestication of pogo-like Transposases into Centromere-Binding Proteins in Fission Yeast and Mammals

The mammalian centromere-associated protein B (CENP-B) shares significant sequence similarity with 3 proteins in fission yeast (Abp1, Cbh1, and Cbh2) that also bind centromeres and have essential function for chromosome segregation and centromeric heterochromatin formation. Each of these proteins displays extensive sequence similarity with pogo-like transposases, which have been previously identified in the genomes of various insects and vertebrates, in the protozoan Entamoeba and in plants. Based on this distribution, it has been proposed that the mammalian and fission yeast centromeric proteins are derived from "domesticated" pogo-like transposons. Here we took advantage of the vast amount of sequence information that has become recently available for a wide range of fungal and animal species to investigate the origin of the mammalian CENP-B and yeast CENP-B-like genes. A highly conserved ortholog of CENP-B was detected in 31 species of mammals, including opossum and platypus, but was absent from all nonmammalian species represented in the databases. Similarly, no ortholog of the fission yeast centromeric proteins was identified in any of the various fungal genomes currently available. In contrast, we discovered a plethora of novel pogo-like transposons in diverse invertebrates and vertebrates and in several filamentous fungi. Phylogenetic analysis revealed that the mammalian and fission yeast CENP-B proteins fall into 2 distinct monophyletic clades, each of which includes a different set of pogo-like transposons. These results are most parsimoniously explained by independent domestication events of pogo-like transposases into centromeric proteins in the mammalian and fission yeast lineages, a case of "convergent domestication." These findings highlight the propensity of transposases to give rise to new host proteins and the potential of transposons as sources of genetic innovation.

The JmjC domain protein Epe1 prevents unregulated assembly and disassembly of heterochromatin

Heterochromatin normally has prescribed chromosomal positions and must not encroach on adjacent regions. We demonstrate that the fission yeast protein Epe1 stabilises silent chromatin, preventing the oscillation of heterochromatin domains. Epe1 loss leads to two contrasting phenotypes: alleviation of silencing within heterochromatin and expansion of silent chromatin into neighbouring euchromatin. Thus, we propose that Epe1 regulates heterochromatin assembly and disassembly, thereby affecting heterochromatin integrity, centromere function and chromosome segregation fidelity. Epe1 regulates the extent of heterochromatin domains at the level of chromatin, not via the RNAi pathway. Analysis of an ectopically silenced site suggests that heterochromatin oscillation occurs in the absence of heterochromatin boundaries. Epe1 requires predicted iron- and 2-oxyglutarate (2-OG)-binding residues for in vivo function, indicating that it is probably a 2-OG/Fe(II)-dependent dioxygenase. We suggest that, rather than being a histone demethylase, Epe1 may be a protein hydroxylase that affects the stability of a heterochromatin protein, or protein–protein interaction, to regulate the extent of heterochromatin domains. Thus, Epe1 ensures that heterochromatin is restricted to the domains to which it is targeted by RNAi.

Post-translational modifications of Abutilon mosaic virus movement protein (BC1) in fission yeast

The movement protein (MP) of Abutilon mosaic virus (AbMV, Geminiviridae) exhibited a complex band pattern upon gel electrophoresis indicating its post-translational modification when expressed in AbMV-infected plants or, ectopically, in fission yeasts. High-resolution separation according to charge and molecular weight in acetic acid/urea polyacrylamide or sodium dodecyl sulphate polyacrylamide gels followed by western blot analysis revealed a pattern of AbMV MP from infected plants more related to that from fission yeast than from bacteria. For this reason, expression in fission yeast was established as an experimental system to study post-translational modifications of AbMV MP. Metabolic labelling with 32P-orthophosphate confirmed a phosphorylation of all MP variants suggesting multiple phosphorylation sites. Treatment with calf intestinal alkaline phosphatase removed this label completely, but was unable to eliminate all protein bands with lower electrophoretic mobility. Thus, multiple phosphorylations contribute to the complex migration behaviour of MP, but additional post-translational modifications may occur as well.

Activation of MPF in fission yeast.

In fission yeast p34cdc2/cyclin is activated at the G2/M boundary by dephosphorylation of Tyr15 of the p34cdc2 subunit. Two protein phosphatases carry out this dephosphorylation event. The major activity is encoded by cdc25, which is a distantly related member of the protein tyrosine phosphatase family. A minor activity is provided by a newly identified fission yeast protein tyrosine phosphatase.

Anchoring microtubules at the spindle poles

In the cell, microtubule organizing centers (MTOCs) dynamically nucleate microtubules and arrange them in functional patterns, but microtubule anchoring to MTOCs is not well understood. A novel fission-yeast protein that anchors the γ-tubulin-containing nucleating complex (γ-TuC) at the spindle pole during mitosis has now been described. The work highlights the complex regulation of microtubule anchoring.

Chromosome Congression: The Kinesin-8-Step Path to Alignment

During mitosis, chromosomes must become aligned at the equator of the mitotic spindle before segregation. Recent work suggests that a kinesin-8 motor uses a unique combination of activities to regulate this process.

A Proteomic Study of Clp1‐interacting Proteins in Fission Yeast

A Proteomic Study of Clp1‐interacting Proteins in Fission Yeast

2‐oxoglutarate‐Fe(II)‐dependent dioxygenase regulates degradation of nuclear sterol regulatory element binding protein in fission yeast

2‐oxoglutarate‐Fe(II)‐dependent dioxygenase regulates degradation of nuclear sterol regulatory element binding protein in fission yeast

Genetic screen to identify regulators of nuclear sterol regulatory element binding protein (SREBP) in fission yeast

Genetic screen to identify regulators of nuclear sterol regulatory element binding protein (SREBP) in fission yeast

Oxygen‐dependent regulation of sterol regulatory element binding protein (SREBP) in fission yeast

Sre1, the fission yeast Sterol Regulatory Element Binding Protein (SREBP), is a principal regulator of low oxygen gene expression whose activation is required for low oxygen growth. Oxygen regulates the activity of Sre1 by two independent mechanisms: Sre1 proteolysis is stimulated by inhibition of oxygen-dependent sterol synthesis; and oxygen accelerates the degradation of nuclear Sre1. Here, we report the identification of 4-methyl sterols as a signal for activation of Sre1 proteolysis. These sterols accumulate under low oxygen and activate Sre1 processing when added exogenously. In addition, we describe the identification of an uncharacterized 2-oxoglutarate, iron-dependent dioxygenase that is required for the oxygen-dependent degradation of nuclear Sre1. This bivalent regulation of Sre1 permits the rapid activation of gene expression and adaptation to fluctuations in environmental oxygen. These studies have important implications for our understanding of sterol-sensing domain proteins and for regulation of SREBP degradation in mammalian cells. Research was funded by a grant from the NIH HL-077588, a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (PJE), and American Heart Association Predoctoral Fellowships (ALH and BTH).

Regulation of the Nuclear Poly(A)-binding Protein by Arginine Methylation in Fission Yeast

Two structurally different poly(A)-binding proteins (PABP) bind the poly(A) tract of mRNAs in most mammalian cells: PABPC in the cytoplasm and PABP2/PABPN1 in the nucleus. Whereas yeast orthologs of the cytoplasmic PABP are characterized, a gene product homologous to mammalian PABP2 has not been identified in yeast. We report here the identification of a homolog of PABP2 as an arginine methyltransferase 1 (RMT1)-associated protein in fission yeast. The product of the Schizosaccharomyces pombe pab2 gene encodes a nonessential nuclear protein and demonstrates specific poly(A) binding in vitro. Consistent with a functional role in poly(A) tail metabolism, mRNAs from pab2-null cells displayed hyperadenylated 3'-ends. We also show that arginine residues within the C-terminal arginine-rich domain of Pab2 are modified by RMT1-dependent methylation. Whereas the arginine methylated and unmethylated forms of Pab2 behaved similarly in terms of subcellular localization, poly(A) binding, and poly(A) tail length control; Pab2 oligomerization levels were markedly increased when Pab2 was not methylated. Significantly, Pab2 overexpression reduced growth rate, and this growth inhibitory effect was exacerbated in rmt1-null cells. Our results indicate that the main cellular function of Pab2 is in poly(A) tail length control and support a biological role for arginine methylation in the regulation of Pab2 oligomerization.

RNA Degradation in Fission Yeast Mitochondria Is Stimulated by a Member of a New Family of Proteins that Are Conserved in Lower Eukaryotes

We report here on the role of open reading frame (ORF) SPCC1183.04c of Schizosaccharomyces pombe in mitochondrial RNA metabolism. A mutant deleted for this ORF on chromosome III accumulates mitochondrial transcripts with the exception of the cob mRNA. A detailed Northern blot analysis showed that the effect results from a decrease in RNA degradation but not from RNA processing deficiencies. Overexpression of the SPCC1183.04c gene in a S. pombe wild-type strain is characterized by slow growth at 37 °C on non-fermentable carbon sources and a significant reduction of steady-state levels of mitochondrial transcripts. A NCBI BLASTP search with the amino acid sequence deduced from the S. pombe gene identified significant similarity to a number of proteins in fungi (e.g. Ascomycota, Basidiomycota) and in some non-fungal eukaryotes (e.g. ciliate, slime mold, red algae). By heterologous expression of SPCC1183.04c in a Saccharomyces cerevisiae pet127Δ strain, we demonstrate that the fission yeast protein and Pet127p from S. cerevisiae function similarly: The fission yeast gene complemented the respiratory defect associated with the pet127Δ allele and partially restored the RNA processing phenotype. Although it lacks any recognizable targeting signal, the S. pombe protein is imported into S. cerevisiae mitochondria in vivo. We conclude from our results that the fission yeast SPCC1183.04c gene is a member of a new protein family that functions to stimulate mitochondrial RNA degradation, a function that is conserved within the mitochondria of lower eukaryotes but seems to have been replaced by alternative pathways in metazoans and higher plants.

Comparative proteomic and transcriptomic profiling of the fission yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a widely used model organism to study basic mechanisms of eukaryotic biology, but unlike other model organisms, its proteome remains largely uncharacterized. Using a shotgun proteomics approach based on multidimensional prefractionation and tandem mass spectrometry, we have detected ∼30% of the theoretical fission yeast proteome. Applying statistical modelling to normalize spectral counts to the number of predicted tryptic peptides, we have performed label-free quantification of 1465 proteins. The fission yeast protein data showed considerable correlations with mRNA levels and with the abundance of orthologous proteins in budding yeast. Functional pathway analysis indicated that the mRNA–protein correlation is strong for proteins involved in signalling and metabolic processes, but increasingly discordant for components of protein complexes, which clustered in groups with similar mRNA–protein ratios. Self-organizing map clustering of large-scale protein and mRNA data from fission and budding yeast revealed coordinate but not always concordant expression of components of functional pathways and protein complexes. This finding reaffirms at the protein level the considerable divergence in gene expression patterns of the two model organisms that was noticed in previous transcriptomic studies.