A conserved non-canonical docking mechanism regulates the binding of dual specificity phosphatases to cell integrity mitogen-activated protein kinases (MAPKs) in budding and fission yeasts.

Dual-specificity MAPK phosphatases (MKPs) are essential for the negative regulation of MAPK pathways. Similar to other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains known as D-motifs. However, we found that the Saccharomyces cerevisiae MKP Msg5 binds the MAPK Slt2 within the cell wall integrity (CWI) pathway through a distinct motif (IYT). Here, we demonstrate that the IYT motif mediates binding of the Msg5 paralogue Sdp1 to Slt2 as well as of the MKP Pmp1 to its CWI MAPK counterpart Pmk1 in the evolutionarily distant yeast Schizosaccharomyces pombe. As a consequence, removal of the IYT site in Msg5, Sdp1 and Pmp1 reduces MAPK trapping caused by the overexpression of catalytically inactive versions of these phosphatases. Accordingly, an intact IYT site is necessary for inactive Sdp1 to prevent nuclear accumulation of Slt2. We also show that both Ile and Tyr but not Thr are essential for the functionality of the IYT motif. These results provide mechanistic insight into MKP-MAPK interplay and stress the relevance of this conserved non-canonical docking site in the regulation of the CWI pathway in fungi.

Crystal structure of the C17/25 subcomplex from Schizosaccharomyces pombe RNA polymerase III.

Eukaryotic RNA polymerase III (Pol III) is a multisubunit enzyme responsible for transcribing tRNA, 5S rRNA, and several small RNAs. Of the 17 subunits in Pol III, the C17 (Rpc17) and C25 (Rpc25) subunits form a stable subcomplex that protrudes from the core polymerase. In this study, we determined the crystal structure of the C17/25 subcomplex from Schizosaccharomyces pombe. The subcomplex adopts an elongated shape, and each subunit has two domains. The two subunits in the subcomplex are tightly packed and extensively interact, with a contact area of 2080 Å(2) . The overall conformation of S. pombe C17/25 is considerably different from the previously reported structure of C17/25 from Saccharomyces cerevisiae, with respect to the position of the C17 HRDC domain, a helix bundle essential for cell viability. In contrast, the S. pombe C17/25 structure is quite similar to those of the Pol II and archaeal counterparts, Rpb4/7 and RpoE/F, respectively, despite the low sequence similarity. A phylogenetic comparison of the C17 subunits among eukaryotes revealed that they can be classified into three groups, according to the length of the interdomain linker. S. pombe C17, as well as Rpb4 and RpoF, belongs to the largest group, with the short linker. On the other hand, S. cerevisiae C17 belongs to the smallest group, with the long linker, which probably enables the subcomplex to assume the alternative conformation.

Two fission yeast rab7 homologs, ypt7 and ypt71, play antagonistic roles in the regulation of vacuolar morphology.

Small guanine triphosphatases (GTPases) of the Rab family are key regulators of membrane trafficking events between the various subcellular compartments in eukaryotic cells. Rab7 is a conserved protein required in the late endocytic pathway and in lysosome biogenesis. A Schizosaccharomyces pombe (S. pombe) homolog of Rab7, Ypt7, is necessary for trafficking from the endosome to the vacuole and for homotypic vacuole fusion. Here, we identified and characterized a second fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to the vacuolar membrane. Cells deleted for ypt71(+) exhibit normal growth rates and morphology. Interestingly, a ypt71 null mutant contains large vacuoles in contrast with the small fragmented vacuoles found in the ypt7 null mutant. Furthermore, the ypt71 mutation does not enhance or alleviate the temperature sensitivity or vacuole fusion defect of ypt7Delta cells. Like ypt7Delta cells, overexpression of ypt71(+) caused fragmentation of vacuoles and inhibits vacuole fusion under hypotonic conditions. Thus, the two S. pombe Rab7 homologs act antagonistically in regulating vacuolar morphology. Analysis of a chimeric Ypt7/Ypt71 protein showed that Rab7-directed vacuole dynamics, fusion versus fission, largely depends on the medial region of the protein, including a part of RabSF3/alpha3-L7.

Identification of the fnx1+ and fnx2+ genes for vacuolar amino acid transporters in Schizosaccharomyces pombe.

We have identified the Schizosaccharomyces pombe SPBC3E7.06c gene (fnx2(+)) from a homology search with the fnx1(+) gene involving in G(0) arrest upon nitrogen starvation. Green fluorescent protein-fused Fnx1p and Fnx2p localized exclusively to the vacuolar membrane. Uptake of histidine or isoleucine by S. pombe cells was inhibited by concanamycin A, a specific inhibitor of the vacuolar H(+)-ATPase. Amino acid uptake was also defective in the vacuolar ATPase mutant, suggesting that vacuolar compartmentalization is critical for amino acid uptake by whole cells. In both Deltafnx1 and Deltafnx2 mutant cells, uptake of lysine, isoleucine or asparagine was impaired. These results suggest that fnx1(+) and fnx2(+) are involved in vacuolar amino acid uptake in S. pombe.

Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solfataricus P2.

The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins. However, it is not known whether these SsoCdc6 proteins can functionally interact and collectively contribute to DNA replication initiation. In the current work, we found that SsoCdc6-1 stimulates DNA-binding activities of SsoCdc6-3. In contrast, SsoCdc6-3 inhibits those of both SsoCdc6-1 and SsoCdc6-2. These regulatory functions are differentially affected by the C-terminal domains of these SsoCdc6 proteins. These data, in conjunction with studies on physical interactions between these replication initiators by bacterial two-hybrid and pull-down/Western blot assays, lead us to propose the possibility that multiple SsoCdc6 proteins might coordinately regulate DNA replication in the archaeon species. This is the first report on the functional interaction among the archaeal multiple Cdc6 proteins to regulate DNA replication.

Proteome analysis of Schizosaccharomyces pombe by two-dimensional gel electrophoresis and mass spectrometry.

The fission yeast Schizosaccharomyces pombe (S. pombe) is a unicellular eukaryote and contains many genes and regulatory mechanisms that are close to those of mammals. In this study, we performed a global proteomic analysis of the fission yeast S. pombe wild type h(-S) L 972 proteome. More than 1,500 protein spots were visualized on silver stained 2-D gels in the 3-10 pI range with a high resolution and high reproducibility. Protein identification was carried out by MALDI-TOF-MS and/or nanoLC-MS/MS. Advantage of the complementarity of these two MS approaches was used to enhance the identification quality. So far, 364 proteins (representing 157 different proteins) have been identified. We report here the identification of 117 new proteins on our 2-D reference map of this yeast compared to the first reference map. Of these identified proteins, 40.1% were involved in metabolism. The present work provides a very useful tool for all studies relying on S. pombe as a model organism and is a considerable complement to the first reference map of S. pombe published recently by Sun and coworkers (Sun, N., Jang, J., Lee, S., Kim, S. et al.., Proteomics 2005, 5, 1574-1579).

The homologous putative GTPases Grn1p from fission yeast and the human GNL3L are required for growth and play a role in processing of nucleolar pre-rRNA.

Grn1p from fission yeast and GNL3L from human cells, two putative GTPases from the novel HSR1_MMR1 GTP-binding protein subfamily with circularly permuted G-motifs play a critical role in maintaining normal cell growth. Deletion of Grn1 resulted in a severe growth defect, a marked reduction in mature rRNA species with a concomitant accumulation of the 35S pre-rRNA transcript, and failure to export the ribosomal protein Rpl25a from the nucleolus. Deleting any of the Grn1p G-domain motifs resulted in a null phenotype and nuclear/nucleolar localization consistent with the lack of nucleolar export of preribosomes accompanied by a distortion of nucleolar structure. Heterologous expression of GNL3L in a Deltagrn1 mutant restored processing of 35S pre-rRNA, nuclear export of Rpl25a and cell growth to wild-type levels. Genetic complementation in yeast and siRNA knockdown in HeLa cells confirmed the homologous proteins Grn1p and GNL3L are required for growth. Failure of two similar HSR1_MMR1 putative nucleolar GTPases, Nucleostemin (NS), or the dose-dependent response of breast tumor autoantigen NGP-1, to rescue deltagrn1 implied the highly specific roles of Grn1p or GNL3L in nucleolar events. Our analysis uncovers an important role for Grn1p/GNL3L within this unique group of nucleolar GTPases.

A Rho-type GTPase, rho-4, is required for septation in Neurospora crassa.

Proteins in the Rho family are small monomeric GTPases primarily involved in polarization, control of cell division, and reorganization of cytoskeletal elements. Phylogenetic analysis of predicted fungal Rho proteins suggests that a new Rho-type GTPase family, whose founding member is Rho4 from the archiascomycete Schizosaccharomyces pombe, is involved in septation. S. pombe rho4Delta mutants have multiple, abnormal septa. In contrast to S. pombe rho4Delta mutants, we show that strains containing rho-4 loss-of-function mutations in the filamentous fungus Neurospora crassa lead to a loss of septation. Epitope-tagged RHO-4 localized to septa and to the plasma membrane. In other fungi, the steps required for septation include formin, septin, and actin localization followed by cell wall synthesis and the completion of septation. rho-4 mutants were unable to form actin rings, showing that RHO-4 is required for actin ring formation. Characterization of strains containing activated alleles of rho-4 showed that RHO-4-GTP is likely to initiate new septum formation in N. crassa.

Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe.

As predicted by the amino acid sequence, the purified protein coded by Schizosaccharomyces pombe SPAC2F7.06c is a DNA polymerase (SpPol4) whose biochemical properties resemble those of other X family (PolX) members. Thus, this new PolX is template-dependent, polymerizes in a distributive manner, lacks a detectable 3'-->5' proofreading activity and its preferred substrates are small gaps with a 5'-phosphate group. Similarly to Polmu, SpPol4 can incorporate a ribonucleotide (rNTP) into a primer DNA. However, it is not responsible for the 1-2 rNTPs proposed to be present at the mating-type locus and those necessary for mating-type switching. Unlike Polmu, SpPol4 lacks terminal deoxynucleotidyltransferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less error-prone than Polmu. Nonetheless, the biochemical properties of this gap-filling DNA polymerase are suitable for a possible role of SpPol4 in non-homologous end-joining. Unexpectedly based on sequence analysis, SpPol4 has deoxyribose phosphate lyase activity like Polbeta and Pollambda, and unlike Polmu, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzymatic properties are hybrid of those described for mammalian Polbeta, Pollambda and Polmu.

Effects of {gamma}-tubulin complex proteins on microtubule nucleation and catastrophe in fission yeast.

Although gamma-tubulin complexes (gamma-TuCs) are known as microtubule (MT) nucleators, their function in vivo is still poorly defined. Mto1p (also known as mbo1p or mod20p) is a gamma-TuC-associated protein that recruits gamma-TuCs specifically to cytoplasmic MT organizing centers (MTOCs) and interphase MTs. Here, we investigated gamma-TuC function by analyzing MT behavior in mto1Delta and alp4 (GCP2 homologue) mutants. These cells have free, extra-long interphase MTs that exhibit abnormal behaviors such as cycles of growth and breakage, MT sliding, treadmilling, and hyperstability. The plus ends of interphase and spindle MTs grow continuously, exhibiting catastrophe defects that are dependent on the CLIP170 tip1p. The minus ends of interphase MTs exhibit shrinkage and pauses. As mto1Delta mutants lack cytoplasmic MTOCs, cytoplasmic MTs arise from spindle or other intranuclear MTs that exit the nucleus. Our findings show that mto1p and gamma-TuCs affect multiple properties of MTs including nucleation, nuclear attachment, plus-end catastrophe, and minus-end shrinkage.

Ase1p organizes antiparallel microtubule arrays during interphase and mitosis in fission yeast.

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Delta mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.

Functional comparison of the Tup11 and Tup12 transcriptional corepressors in fission yeast.

Gene duplication is considered an important evolutionary mechanism. Unlike many characterized species, the fission yeast Schizosaccharomyces pombe contains two paralogous genes, tup11+ and tup12+, that encode transcriptional corepressors similar to the well-characterized budding yeast Tup1 protein. Previous reports have suggested that Tup11 and Tup12 proteins play redundant roles. Consistently, we show that the two Tup proteins can interact together when expressed at normal levels and that each can independently interact with the Ssn6 protein, as seen for Tup1 in budding yeast. However, tup11- and tup12- mutants have different phenotypes on media containing KCl and CaCl2. Consistent with the functional difference between tup11- and tup12- mutants, we identified a number of genes in genome-wide gene expression experiments that are differentially affected by mutations in the tup11+ and tup12+ genes. Many of these genes are differentially derepressed in tup11- mutants and are over-represented in genes that have previously been shown to respond to a range of different stress conditions. Genes specifically derepressed in tup12- mutants require the Ssn6 protein for their repression. As for Tup12, Ssn6 is also required for efficient adaptation to KCl- and CaCl2-mediated stress. We conclude that Tup11 and Tup12 are at least partly functionally diverged and suggest that the Tup12 and Ssn6 proteins have adopted a specific role in regulation of the stress response.

A description of the Mei2-like protein family; structure, phylogenetic distribution and biological context.

The Schizosaccharomyces pombe Mei2 gene encodes an RNA recognition motif (RRM) protein that stimulates meiosis upon binding a specific non-coding RNA and subsequent accumulation in a "mei2-dot" in the nucleus. We present here the first systematic characterization of the family of proteins with characteristic Mei2-like amino acid sequences. Mei2-like proteins are an ancient eukaryotic protein family with three identifiable RRMs. The C-terminal RRM (RRM3) is unique to Mei2-like proteins and is the most highly conserved of the three RRMs. RRM3 also contains conserved sequence elements at its C-terminus not found in other RRM domains. Single copy Mei2-like genes are present in some fungi, in alveolates such as Paramecium and in the early branching eukaryote Entamoeba histolytica, while plants contain small families of Mei2-like genes. While the C-terminal RRM is highly conserved between plants and fungi, indicating conservation of molecular mechanisms, plant Mei2-like genes have changed biological context to regulate various aspects of developmental pattern formation.

A fourth component of the fission yeast gamma-tubulin complex, Alp16, is required for cytoplasmic microtubule integrity and becomes indispensable when gamma-tubulin function is compromised.

gamma-Tubulin functions as a multiprotein complex, called the gamma-tubulin complex (gamma-TuC), and composes the microtubule organizing center (MTOC). Fission yeast Alp4 and Alp6 are homologues of two conserved gamma-TuC proteins, hGCP2 and hGCP3, respectively. We isolated a novel gene, alp16(+), as a multicopy suppressor of temperature-sensitive alp6-719 mutants. alp16(+) encodes a 759-amino-acid protein with two conserved regions found in all other members of gamma-TuC components. In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210. Gene disruption shows that alp16(+) is not essential for cell viability. However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip. Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225, alp4-1891, or alp6-719 mutants. Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6. Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase. Alp16 coimmunoprecipitates with gamma-tubulin and cosediments with the gamma-TuC in a large complex (>20 S). Alp16 is, however, not required for the formation of this large complex. We discuss evolutional conservation and divergence of structure and function of the gamma-TuC between yeast and higher eukaryotes.