Dynein motion switches from diffusive to directed upon cortical anchoring.

Cytoplasmic dynein is a motor protein that exerts force on microtubules. To generate force for the movement of large organelles, dynein needs to be anchored, with the anchoring sites being typically located at the cell cortex. However, the mechanism by which dyneins target sites where they can generate large collective forces is unknown. Here, we directly observe single dyneins during meiotic nuclear oscillations in fission yeast and identify the steps of the dynein binding process: from the cytoplasm to the microtubule and from the microtubule to cortical anchors. We observed that dyneins on the microtubule move either in a diffusive or directed manner, with the switch from diffusion to directed movement occurring upon binding of dynein to cortical anchors. This dual behavior of dynein on the microtubule, together with the two steps of binding, enables dyneins to self-organize into a spatial pattern needed for them to generate large collective forces.

Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells.

Data on absolute molecule numbers will empower the modeling, understanding, and comparison of cellular functions and biological systems. We quantified transcriptomes and proteomes in fission yeast during cellular proliferation and quiescence. This rich resource provides the first comprehensive reference for all RNA and most protein concentrations in a eukaryote under two key physiological conditions. The integrated data set supports quantitative biology and affords unique insights into cell regulation. Although mRNAs are typically expressed in a narrow range above 1 copy/cell, most long, noncoding RNAs, except for a distinct subset, are tightly repressed below 1 copy/cell. Cell-cycle-regulated transcription tunes mRNA numbers to phase-specific requirements but can also bring about more switch-like expression. Proteins greatly exceed mRNAs in abundance and dynamic range, and concentrations are regulated to functional demands. Upon transition to quiescence, the proteome changes substantially, but, in stark contrast to mRNAs, proteins do not uniformly decrease but scale with cell volume.

Label-Free Quantitative Phosphoproteomics of the Fission Yeast Schizosaccharomyces pombe Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies.

The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast Schizosaccharomyces pombe. Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the S. pombe phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast S. pombe.

Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells.

During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal in vivo In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast (Schizosaccharomyces pombe). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing in vivo The mean and median lengths of the flaps in wild-type cells were ∼51 and ∼41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided direct in vivo evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish a new strategy for interrogation of this fascinating process.

Fission yeast Scm3 mediates stable assembly of Cnp1/CENP-A into centromeric chromatin.

Mis16 and Mis18 are subunits of a protein complex required for incorporation of the histone H3 variant CenH3 (Cnp1/CENP-A) into centromeric chromatin in Schizosaccharomyces pombe and mammals. How the Mis16-Mis18 complex performs this function is unknown. Here, we report that the Mis16-Mis18 complex is required for centromere localization of Scm3(Sp), a Cnp1-binding protein related to Saccharomyces cerevisiae Scm3. Scm3(Sp) is required for centromeric localization of Cnp1, while Scm3(Sp) localizes at centromeres independently of Cnp1. Like the Mis16-Mis18 complex but unlike Cnp1, Scm3(Sp) dissociates from centromeres during mitosis. Inactivation of Scm3(Sp) or Mis18 increases centromere localization of histones H3 and H2A/H2B, which are largely absent from centromeres in wild-type cells. Whereas S. cerevisiae Scm3 is proposed to replace histone H2A/H2B in centromeric nucleosomes, the dynamic behavior of S. pombe Scm3 suggests that it acts as a Cnp1 assembly/maintenance factor that directly mediates the stable deposition of Cnp1 into centromeric chromatin.

Fission yeast Scm3: A CENP-A receptor required for integrity of subkinetochore chromatin.

The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

The eIF3 interactome reveals the translasome, a supercomplex linking protein synthesis and degradation machineries.

eIF3 promotes translation initiation, but relatively little is known about its full range of activities in the cell. Here, we employed affinity purification and highly sensitive LC-MS/MS to decipher the fission yeast eIF3 interactome, which was found to contain 230 proteins. eIF3 assembles into a large supercomplex, the translasome, which contains elongation factors, tRNA synthetases, 40S and 60S ribosomal proteins, chaperones, and the proteasome. eIF3 also associates with ribosome biogenesis factors and the importins-beta Kap123p and Sal3p. Our genetic data indicated that the binding to both importins-beta is essential for cell growth, and photobleaching experiments revealed a critical role for Sal3p in the nuclear import of one of the translasome constituents, the proteasome. Our data reveal the breadth of the eIF3 interactome and suggest that factors involved in translation initiation, ribosome biogenesis, translation elongation, quality control, and transport are physically linked to facilitate efficient protein synthesis.

Unraveling Site-Specific and Combinatorial Histone Modifications Using High-Resolution Mass Spectrometry in Histone Deacetylase Mutants of Fission Yeast.

Histone deacetylases (HDACs) catalyze the removal of acetylation marks from lysine residues on histone and nonhistone substrates. Their activity is generally associated with essential cellular processes such as transcriptional repression and heterochromatin formation. Interestingly, abnormal activity of HDACs has been reported in various types of cancers, which makes them a promising therapeutic target for cancer treatment. In the current study, we aim to understand the mechanisms underlying the function of HDACs using an in-depth quantitative analysis of changes in histone acetylation levels in Schizosaccharomyces pombe (S. pombe) lacking major HDAC activities. We employed a targeted quantitative mass spectrometry approach to profile changes of acetylation and methylation at multiple lysine residues on the N-terminal tail of histones H3 and H4. Our analyses identified a number of histone acetylation sites that are significantly affected by S. pombe HDAC mutations. We discovered that mutation of the Class I HDAC known as Clr6 causes a major increase in the abundance of triacetylated H4 molecules at K5, K8, and K12. A clr6-1 hypomorphic mutation also increased the abundance of multiple acetyl-lysines in histone H3. In addition, our study uncovered a few crosstalks between histone acetylation and methylation upon deletion of HDACs Hos2 and Clr3. We anticipate that the results from this study will greatly improve our current understanding of the mechanisms involved in HDAC-mediated gene regulation and heterochromatin assembly.

Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity.

Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1-Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking.

Absolute proteome and phosphoproteome dynamics during the cell cycle of Schizosaccharomyces pombe (Fission Yeast).

To quantify cell cycle-dependent fluctuations on a proteome-wide scale, we performed integrative analysis of the proteome and phosphoproteome during the four major phases of the cell cycle in Schizosaccharomyces pombe. In highly synchronized cells, we identified 3753 proteins and 3682 phosphorylation events and relatively quantified 65% of the data across all phases. Quantitative changes during the cell cycle were infrequent and weak in the proteome but prominent in the phosphoproteome. Protein phosphorylation peaked in mitosis, where the median phosphorylation site occupancy was 44%, about 2-fold higher than in other phases. We measured copy numbers of 3178 proteins, which together with phosphorylation site stoichiometry enabled us to estimate the absolute amount of protein-bound phosphate, as well as its change across the cell cycle. Our results indicate that 23% of the average intracellular ATP is utilized by protein kinases to phosphorylate their substrates to drive regulatory processes during cell division. Accordingly, we observe that phosphate transporters and phosphate-metabolizing enzymes are phosphorylated and therefore likely to be regulated in mitosis.

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.

A novel factor Iss10 regulates Mmi1-mediated selective elimination of meiotic transcripts.

A number of meiosis-specific transcripts are selectively eliminated during the mitotic cell cycle in fission yeast. Mmi1, an RNA-binding protein, plays a crucial role in this selective elimination. Mmi1 recognizes a specific region, namely, the determinant of selective removal (DSR) on meiotic transcripts and induces nuclear exosome-mediated elimination. During meiosis, Mmi1 is sequestered by a chromosome-associated dot structure, Mei2 dot, allowing meiosis-specific transcripts to be stably expressed. Red1, a zinc-finger protein, is also known to participate in the Mmi1/DSR elimination system, although its molecular function has remained elusive. To uncover the detailed molecular mechanisms underlying the Mmi1/DSR elimination system, we sought to identify factors that interact genetically with Mmi1. Here, we show that one of the identified factors, Iss10, is involved in the Mmi1/DSR system by regulating the interaction between Mmi1 and Red1. In cells lacking Iss10, association of Red1 with Mmi1 is severely impaired, and target transcripts of Mmi1 are ectopically expressed in the mitotic cycle. During meiosis, Iss10 is downregulated, resulting in dissociation of Red1 from Mmi1 and subsequent suppression of Mmi1 activity.

Red5 and three nuclear pore components are essential for efficient suppression of specific mRNAs during vegetative growth of fission yeast.

Zinc-finger domains are found in many nucleic acid-binding proteins in both prokaryotes and eukaryotes. Proteins carrying zinc-finger domains have important roles in various nuclear transactions, including transcription, mRNA processing and mRNA export; however, for many individual zinc-finger proteins in eukaryotes, the exact function of the protein is not fully understood. Here, we report that Red5 is involved in efficient suppression of specific mRNAs during vegetative growth of Schizosaccharomyces pombe. Red5, which contains five C3H1-type zinc-finger domains, localizes to the nucleus where it forms discrete dots. A red5 point mutation, red5-2, results in the upregulation of specific meiotic mRNAs in vegetative mutant red5-2 cells; northern blot data indicated that these meiotic mRNAs in red5-2 cells have elongated poly(A) tails. RNA-fluorescence in situ hybridization results demonstrate that poly(A)(+) RNA species accumulate in the nucleolar regions of red5-deficient cells. Moreover, Red5 genetically interacts with several mRNA export factors. Unexpectedly, three components of the nuclear pore complex also suppress a specific set of meiotic mRNAs. These results indicate that Red5 function is important to meiotic mRNA degradation; they also suggest possible connections among selective mRNA decay, mRNA export and the nuclear pore complex in vegetative fission yeast.

Importance of polyadenylation in the selective elimination of meiotic mRNAs in growing S. pombe cells.

A number of meiosis-specific mRNAs are initially weakly transcribed, but then selectively removed during fission yeast mitotic growth. These mRNAs harbour a region termed DSR (determinant of selective removal), which is recognized by the YTH family RNA-binding protein Mmi1p. Mmi1p directs the destruction of these mRNAs in collaboration with nuclear exosomes. However, detailed molecular mechanisms underlying this process of selective mRNA elimination have remained elusive. In this study, we demonstrate the critical role of polyadenylation in this process. Two-hybrid and genetic screens revealed potential interactions between Mmi1p and proteins involved in polyadenylation. Additional investigations showed that destruction of DSR-containing mRNAs by exosomes required polyadenylation by a canonical poly(A) polymerase. The recruitment of Pab2p, a poly(A)-binding protein, to the poly(A) tail was also necessary for mRNA destruction. In cells undergoing vegetative growth, Mmi1p localized with exosomes, Pab2p, and components of the polyadenylation complex in several patchy structures in the nucleoplasm. These patches may represent the sites for degradation of meiosis-specific mRNAs with untimely expression.

A highly efficient multifunctional tandem affinity purification approach applicable to diverse organisms.

Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions.

Chromatin architectures at fission yeast transcriptional promoters and replication origins.

We have used micrococcal nuclease (MNase) digestion followed by deep sequencing in order to obtain a higher resolution map than previously available of nucleosome positions in the fission yeast, Schizosaccharomyces pombe. Our data confirm an unusually short average nucleosome repeat length, ∼152 bp, in fission yeast and that transcriptional start sites (TSSs) are associated with nucleosome-depleted regions (NDRs), ordered nucleosome arrays downstream and less regularly spaced upstream nucleosomes. In addition, we found enrichments for associated function in four of eight groups of genes clustered according to chromatin configurations near TSSs. At replication origins, our data revealed asymmetric localization of pre-replication complex (pre-RC) proteins within large NDRs-a feature that is conserved in fission and budding yeast and is therefore likely to be conserved in other eukaryotic organisms.

In vivo probing of the temperature responses of intracellular biomolecules in yeast cells by label-free Raman microspectroscopy.

Environmental temperature is an essential physical quantity that substantially influences cell physiology by changing the equilibria and kinetics of biochemical reactions occurring in cells. Although it has been extensively used as a readily controllable parameter in genetic and biochemical research, much remains to be explored about the temperature responses of intracellular biomolecules in vivo and at the molecular level. Here we report in vivo probing, achieved with label-free Raman microspectroscopy, of the temperature responses of major intracellular components such as lipids and proteins in living fission yeast cells. The characteristic Raman band at 1602 cm(-1), which has been attributed mainly to ergosterol, showed a significant decrease (≈47 %) in intensity at elevated temperatures above 35 °C. In contrast to this high temperature sensitivity of the ergosterol Raman band, the phospholipid and protein Raman bands did not vary much with increasing culture temperature in the 26-38 °C range. This finding agrees with a previous biochemical study that showed that the initial stages of ergosterol biosynthesis in yeast are hindered by temperature elevation. Moreover, our result demonstrates that Raman microspectroscopy holds promise for elucidation of temperature-dependent cellular activities in living cells, with a high molecular specificity that the commonly used fluorescence microscopy cannot offer.

A novel function of the mitochondrial transcription factor Mtf1 in fission yeast; Mtf1 regulates the nuclear transcription of srk1.

In eukaryotic cells, Mtf1 and its homologues function as mitochondrial transcription factors for the mitochondrial RNA polymerase in the mitochondrion. Here we show that in fission yeast Mtf1 exerts a non-mitochondrial function as a nuclear factor that regulates transcription of srk1, which is a kinase involved in the stress response and cell cycle progression. We first found Mtf1 expression in the nucleus. A ChIP-chip approach identified srk1 as a putative Mtf1 target gene. Over expression of Mtf1 induced transcription of the srk1 gene and Mtf1 deletion led to a reduction in transcription of the srk1 gene in vivo. Mtf1 overexpression causes cell elongation in a srk1 dependent manner. Mtf1 overexpression can cause cytoplasmic accumulation of Cdc25. We also provide biochemical evidence that Mtf1 binds to the upstream sequence of srk1. This is the first evidence that a mitochondrial transcription factor Mtf1 can regulate a nuclear gene. Mtf1 may also have a role in cell cycle progression.

A failure of meiotic chromosome segregation in a fbh1Delta mutant correlates with persistent Rad51-DNA associations.

The F-box DNA helicase Fbh1 constrains homologous recombination in vegetative cells, most likely through an ability to displace the Rad51 recombinase from DNA. Here, we provide the first evidence that Fbh1 also serves a vital meiotic role in fission yeast to promote normal chromosome segregation. In the absence of Fbh1, chromosomes remain entangled or segregate unevenly during meiosis, and genetic and cytological data suggest that this results in part from a failure to efficiently dismantle Rad51 nucleofilaments that form during meiotic double-strand break repair.

The FN3 and BRCT motifs in the exomer component Chs5p define a conserved module that is necessary and sufficient for its function.

Chs5p is a component of the exomer, a coat complex required to transport the chitin synthase Chs3p from the trans-Golgi network to the plasma membrane. The Chs5p N-terminal region exhibits fibronectin type III (FN3) and BRCT domains. FN3 domains are present in proteins that mediate adhesion processes, whereas BRCT domains are involved in DNA repair. Several fungi--including Schizosaccharomyces pombe, which has no detectable amounts of chitin--have proteins similar to Chs5p. Here we show that the FN3 and BRCT motifs in Chs5p behave as a module that is necessary and sufficient for Chs5p localization and for cargo delivery. The N-terminal regions of S. cerevisiae Chs5p and S. pombe Cfr1p are interchangeable in terms of Golgi localization, but not in terms of exomer assembly, showing that the conserved function of this module is protein retention in this organelle and that the interaction between the exomer components is organism-specific.