Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Hiromi Maekawa Last modified date:2019.06.24

Lecturer / Attached Promotive Center for International Education and Research of Agriculture / Faculty of Agriculture


Papers
1. Seike T, Maekawa H, Nakamura T, Shimoda C., The asymmetric chemical structures of two mating pheromones reflect their differential roles in mating of fission yeast., J. Cell Sci., 2019.06.
2. J. Mala, S. Puthong, Hiromi Maekawa, Y. Kaneko, T. Palaga, K. Komolpis, S. Sooksai, Expression and characterization of functional single-chain variable fragment against norfloxacin in Pichia pastoris GS115, International Food Research Journal, 25, 4, 1726-1732, 2018.01, A functional single-chain variable fragment (scFv) of mouse monoclonal antibody against norfloxacin was expressed in yeast Pichia pastoris GS115, purified and characterized. Gene encoding monoclonal antibody against norfloxacin was transformed to P. pastoris GS115 using the pJM01 plasmid. Integration of the plasmid into the genome was verified by PCR and DNA sequencing analysis. After screening for the transformant and production of the selected scFv, the norfloxacin-binding ability was determined by an enzyme linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis. Eight transformats (O1-O8) were obtained from the transformation of P. pastoris GS115 with PmeI-linearized pJM01. PCR products detected by gel electrophoresis confirmed that pJM01 expression cassette was integrated at AOX1 promoter. After screening all transformants for high expression of the recombinant scFv, transformant O5 was selected for antibody production and purification. Detection of norfloxacin by ELISA with the recombinant scFv fragment was successful albeit with lower sensitivity compared to the original monoclonal antibody. In SPR analysis, our recombinant scFv fragments have the binding capability to norfloxacin equivalent to the original antibody with the average angle shift of 580 ± 133 and 505 ± 28 mDegree, respectively. The functional recombinant scFv of monoclonal antibody against norfloxacin successfully produced by methylotrophic yeast, P. pastoris GS115..
3. Katsuyoshi Yamamoto, Thi N.M. Tran, Kaoru Takegawa, Yoshinobu Kaneko, Hiromi Maekawa, Regulation of mating type switching by the mating type genes and RME1 in Ogataea polymorpha, Scientific Reports, 10.1038/s41598-017-16284-7, 7, 1, 2017.12, Saccharomyces cerevisiae and its closely related yeasts undergo mating type switching by replacing DNA sequences at the active mating type locus (MAT) with one of two silent mating type cassettes. Recently, a novel mode of mating type switching was reported in methylotrophic yeast, including Ogataea polymorpha, which utilizes chromosomal recombination between inverted-repeat sequences flanking two MAT loci. The inversion is highly regulated and occurs only when two requirements are met: haploidy and nutritional starvation. However, links between this information and the mechanism associated with mating type switching are not understood. Here we investigated the roles of transcription factors involved in yeast sexual development, such as mating type genes and the conserved zinc finger protein Rme1. We found that co-presence of mating type a1 and α2 genes was sufficient to prevent mating type switching, suggesting that ploidy information resides solely in the mating type locus. Additionally, RME1 deletion resulted in a reduced rate of switching, and ectopic expression of O. polymorpha RME1 overrode the requirement for starvation to induce MAT inversion. These results suggested that mating type switching in O. polymorpha is likely regulated by two distinct transcriptional programs that are linked to the ploidy and transmission of the starvation signal..
4. Minori Numamoto, Hiromi Maekawa, Yoshinobu Kaneko, Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.06.001, 124, 5, 487-492, 2017.11, The methylotrophic yeast Ogataea polymorpha (syn. Hansenula polymorpha) is an attractive industrial non-conventional yeast showing high thermo-tolerance (up to 50°C) and xylose assimilation. However, genetic manipulation of O. polymorpha is often laborious and time-consuming because it has lower homologous recombination efficiency relative to Saccharomyces cerevisiae. To overcome this disadvantage, we applied the CRISPR/Cas9 system as a powerful genome editing tool in O. polymorpha. In this system, both single guide RNA (sgRNA) and endonuclease Cas9 were expressed by a single autonomously-replicable plasmid and the sgRNA portion could be easily changed by using PCR and In-Fusion cloning techniques. Because the mutation efficiency of the CRISPR/Cas9 system was relatively low when the sgRNA was expressed under the control of the OpSNR6 promoter, the tRNACUG gene was used for sgRNA expression. The editing efficiency of this system ranged from 17% to 71% of transformants in several target genes tested (ADE12, PHO1, PHO11, and PHO84). These findings indicate that genetic manipulation of O. polymorpha will be more convenient and accelerated by using this CRISPR/Cas9 system..
5. Hiromi Maekawa, Annett Neuner, Diana Rüthnick, Elmar Schiebel, Gislene Pereira, Yoshinobu Kaneko, Polo-like kinase Cdc5 regulates Spc72 recruitment to spindle pole body in the methylotrophic yeast ogataea polymorpha, eLife, 10.7554/eLife.24340, 6, 2017.08, Cytoplasmic microtubules (cMT) control mitotic spindle positioning in many organisms, and are therefore pivotal for successful cell division. Despite its importance, the temporal control of cMT formation remains poorly understood. Here we show that unlike the best-studied yeast Saccharomyces cerevisiae, position of pre-anaphase nucleus is not strongly biased toward bud neck in Ogataea polymorpha and the regulation of spindle positioning becomes active only shortly before anaphase. This is likely due to the unstable property of cMTs compared to those in S. cerevisiae. Furthermore, we show that cMT nucleation/anchoring is restricted at the level of recruitment of the γ-tubulin complex receptor, Spc72, to spindle pole body (SPB), which is regulated by the polo-like kinase Cdc5. Additionally, electron microscopy revealed that the cytoplasmic side of SPB is structurally different between G1 and anaphase. Thus, polo-like kinase dependent recruitment of γ-tubulin receptor to SPBs determines the timing of spindle orientation in O. polymorpha..
6. J. Mala, S. Puthong, Hiromi Maekawa, Y. Kaneko, T. Palaga, K. Komolpis, S. Sooksai, Construction and sequencing analysis of scFv antibody fragment derived from monoclonal antibody against norfloxacin (Nor155), Journal of Genetic Engineering and Biotechnology, 10.1016/j.jgeb.2017.02.008, 15, 1, 69-76, 2017.06, Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv) that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding VH and VL regions of monoclonal antibody against norfloxacin (Nor155) were amplified and size of VH and VL fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly4Ser)3 as a linker between VH and VL regions and subcloned into pPICZαA, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1) was confirmed by sequencing analysis. The complementarity determining regions (CDR) I, II, and III of VH and VL were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics..
7. Ying Zhou, Naoya Yuikawa, Hiroki Nakatsuka, Hiromi Maekawa, Satoshi Harashima, Yoichi Nakanishi, Yoshinobu Kaneko, Core regulatory components of the PHO pathway are conserved in the methylotrophic yeast Hansenula polymorpha, Current Genetics, 10.1007/s00294-016-0565-7, 62, 3, 595-605, 2016.08, To gain better understanding of the diversity and evolution of the gene regulation system in eukaryotes, the phosphate signal transduction (PHO) pathway in non-conventional yeasts has been studied in recent years. Here we characterized the PHO pathway of Hansenula polymorpha, which is genetically tractable and distantly related to Saccharomyces cerevisiae and Schizosaccharomyces pombe, in order to get more information for the diversity and evolution of the PHO pathway in yeasts. We generated several pho gene-deficient mutants based on the annotated draft genome of H. polymorpha BY4329. Except for the Hppho2-deficient mutant, these mutants exhibited the same phenotype of repressible acid phosphatase (APase) production as their S. cerevisiae counterparts. Subsequently, Hppho80 and Hppho85 mutants were isolated as suppressors of the Hppho81 mutation and Hppho4 was isolated from Hppho80 and Hppho85 mutants as the sole suppressor of the Hppho80 and Hppho85 mutations. To gain more complete delineation of the PHO pathway in H. polymorpha, we screened for UV-irradiated mutants that expressed APase constitutively. As a result, three classes of recessive constitutive mutations and one dominant constitutive mutation were isolated. Genetic analysis showed that one group of recessive constitutive mutations was allelic to HpPHO80 and that the dominant mutation occurred in the HpPHO81 gene. Epistasis analysis between Hppho81 and the other two classes of recessive constitutive mutations suggested that the corresponding new genes, named PHO51 and PHO53, function upstream of HpPHO81 in the PHO pathway. Taking these findings together, we conclude that the main components of the PHO pathway identified in S. cerevisiae are conserved in the methylotrophic yeast H. polymorpha, even though these organisms separated from each other before duplication of the whole genome. This finding is useful information for the study of evolution of the PHO regulatory system in yeasts..
8. Minori Numamoto, Shota Tagami, Yusuke Ueda, Yusuke Imabeppu, Yu Sasano, Minetaka Sugiyama, Hiromi Maekawa, Satoshi Harashima, Nuclear localization domains of GATA activator Gln3 are required for transcription of target genes through dephosphorylation in Saccharomyces cerevisiae, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2014.12.017, 120, 2, 121-127, 2015.08, The GATA transcription activator Gln3 in the budding yeast (. Saccharomyces cerevisiae) activates transcription of nitrogen catabolite repression (NCR)-sensitive genes. In cells grown in the presence of preferred nitrogen sources, Gln3 is phosphorylated in a TOR-dependent manner and localizes in the cytoplasm. In cells grown in non-preferred nitrogen medium or treated with rapamycin, Gln3 is dephosphorylated and is transported from the cytoplasm to the nucleus, thereby activating the transcription of NCR-sensitive genes. Caffeine treatment also induces dephosphorylation of Gln3 and its translocation to the nucleus and transcription of NCR-sensitive genes. However, the details of the mechanism by which phosphorylation controls Gln3 localization and transcriptional activity are unknown. Here, we focused on two regions of Gln3 with nuclear localization signal properties (NLS-K, and NLS-C) and one with nuclear export signal (NES). We constructed various mutants for our analyses: gln3 containing point mutations in all potential phosphoacceptor sites (Thr-339, Ser-344, Ser-347, Ser-355, Ser-391) in the NLS and NES regions to produce non-phosphorylatable (alanine) or mimic-phosphorylatable (aspartic acid) residues; and deletion mutants. We found that phosphorylation of Gln3 was impaired in all of these mutations and that the aspartic acid substitution mutants showed drastic reduction of Gln3-mediated transcriptional activity despite the fact that the mutations had no effect on nuclear localization of Gln3. Our observations suggest that these regions are required for transcription of target genes presumably through dephosphorylation..
9. Minori Numamoto, Yu Sasano, Masataka Hirasaki, Minetaka Sugiyama, Hiromi Maekawa, Satoshi Harashima, The protein phosphatase Siw14 controls caffeine-induced nuclear localization and phosphorylation of Gln3 via the type 2A protein phosphatases Pph21 and Pph22 in Saccharomyces cerevisiae, Journal of Biochemistry, 10.1093/jb/mvu055, 157, 1, 53-64, 2015.01, The Saccharomyces cerevisiae Siw14, a tyrosine phosphatase involved in the response to caffeine, participates in regulation of the phosphorylation and intracellular localization of Gln3, a GATA transcriptional activator of nitrogen catabolite repression-sensitive genes. In Δsiw14 cells, the phosphorylation level of Gln3 is decreased and the nuclear localization of Gln3 is stimulated by caffeine. However, the mechanism by which Siw14 controls the localization and function of Gln3 remains unclear, although the nuclear localization of Gln3 is known to be induced by activation of the type 2A phosphatases (PP2As) Pph21 and Pph22, and the type 2A-related phosphatase Sit4. In this study, we show that the increased nuclear localization of Gln3 in response to caffeine caused by disruption of the SIW14 gene is dependent on the Sit4 and PP2A phosphatases. We also show that decreased phosphorylation of Gln3 caused by disruption of the SIW14 gene is completely suppressed by deletion of both PPH21 and PPH22, but only partially suppressed by deletion of SIT4. Taking these results together, we conclude that Siw14 functions upstream of Pph21 and Pph22 as an inhibitor of the phosphorylation and localization of Gln3, and that Sit4 acts independently of Siw14..
10. Hiromi Maekawa, Yoshinobu Kaneko, Inversion of the Chromosomal Region between Two Mating Type Loci Switches the Mating Type in Hansenula polymorpha, PLoS Genetics, 10.1371/journal.pgen.1004796, 10, 11, 2014.01, Yeast mating type is determined by the genotype at the mating type locus (MAT). In homothallic (self-fertile) Saccharomycotina such as Saccharomyces cerevisiae and Kluveromyces lactis, high-efficiency switching between a and α mating types enables mating. Two silent mating type cassettes, in addition to an active MAT locus, are essential components of the mating type switching mechanism. In this study, we investigated the structure and functions of mating type genes in H. polymorpha (also designated as Ogataea polymorpha). The H. polymorpha genome was found to harbor two MAT loci, MAT1 and MAT2, that are ∼18 kb apart on the same chromosome. MAT1-encoded α1 specifies α cell identity, whereas none of the mating type genes were required for a identity and mating. MAT1-encoded α2 and MAT2-encoded a1 were, however, essential for meiosis. When present in the location next to SLA2 and SUI1 genes, MAT1 or MAT2 was transcriptionally active, while the other was repressed. An inversion of the MAT intervening region was induced by nutrient limitation, resulting in the swapping of the chromosomal locations of two MAT loci, and hence switching of mating type identity. Inversion-deficient mutants exhibited severe defects only in mating with each other, suggesting that this inversion is the mechanism of mating type switching and homothallism. This chromosomal inversion-based mechanism represents a novel form of mating type switching that requires only two MAT loci..
11. Yasushi Hiraoka, Hiromi Maekawa, Haruhiko Asakawa, Yuji Chikashige, Tomoko Kojidani, Hiroko Osakada, Atsushi Matsuda, Tokuko Haraguchi, Inner nuclear membrane protein Ima1 is dispensable for intranuclear positioning of centromeres, Genes to Cells, 10.1111/j.1365-2443.2011.01544.x, 16, 10, 1000-1011, 2011.10, Inner nuclear membrane (INM) proteins play a role in spatial organization of chromosomes within the nucleus. In the fission yeast Schizosaccharomyces pombe, Sad1, an INM protein of the conserved SUN-domain family, plays an active role in moving chromosomes along the nuclear membranes during meiotic prophase. Ima1 is another conserved INM protein recently identified. A previous study claimed that Ima1 is essential for mitotic cell growth, linking centromeric heterochromatin to the spindle-pole body. However, we obtained results contradictory to the previously proposed role for Ima1: Ima1 was dispensable for mitotic cell growth or centromere positioning. This discrepancy was attributed to incorrect ima1 deletion mutants used in the previous study. Our results show that Ima1 collaborates with two other conserved INM proteins of the LEM-domain family that are homologous to human Man1 and Lem2. Loss of any one of three INM proteins has no effect on mitotic cell growth; however, loss of all these proteins causes severe defects in mitotic cell growth and nuclear membrane morphology. Considering that all three INM proteins interact with Sad1, these results suggest that Ima1, Lem2 and Man1 play at least partially redundant roles for nuclear membrane organization..
12. Cornelia König, Hiromi Maekawa, Elmar Schiebel, Mutual regulation of cyclin-dependent kinase and the mitotic exit network, Journal of Cell Biology, 10.1083/jcb.200911128, 188, 3, 351-368, 2010.02, The mitotic exit network (MEN) is a spindle pole body (SPB)-associated, GTPase-driven signaling cascade that controls mitotic exit. The inhibitory Bfa1-Bub2 GTPase-activating protein (GAP) only associates with the daughter SPB (dSPB), raising the question as to how the MEN is regulated on the mother SPB (mSPB). Here, we show mutual regulation of cyclin-dependent kinase 1 (Cdk1) and the MEN. In early anaphase Cdk1 becomes recruited to the mSPB depending on the activity of the MEN kinase Cdc15. Conversely, Cdk1 negatively regulates binding of Cdc15 to the mSPB. In addition, Cdk1 phosphorylates the Mob1 protein to inhibit the activity of Dbf2-Mob1 kinase that regulates Cdc14 phosphatase. Our data revise the understanding of the spatial regulation of the MEN. Although MEN activity in the daughter cells is controlled by Bfa1-Bub2, Cdk1 inhibits MEN activity at the mSPB. Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects..
13. Hiromi Maekawa, Claire Priest, Johannes Lechner, Gislene Pereira, Elmar Schiebel, The yeast centrosome translates the positional information of the anaphase spindle into a cell cycle signal, Journal of Cell Biology, 10.1083/jcb.200705197, 179, 3, 423-436, 2007.11, The spindle orientation checkpoint (SPOC) of budding yeast delays mitotic exit when cytoplasmic microtubules (MTs) are defective, causing the spindle to become misaligned. Delay is achieved by maintaining the activity of the Bfa1-Bub2 guanosine triphosphatase-activating protein complex, an inhibitor of mitotic exit. In this study, we show that the spindle pole body (SPB) component Spc72, a transforming acidic coiled coil-like molecule that interacts with the γ-tubulin complex, recruits Kin4 kinase to both SPBs when cytoplasmic MTs are defective. This allows Kin4 to phosphorylate the SPB-associated Bfa1, rendering it resistant to inactivation by Cdc5 polo kinase. Consistently, forced targeting of Kin4 to both SPBs delays mitotic exit even when the anaphase spindle is correctly aligned. Moreover, we present evidence that Spc72 has an additional function in SPOC regulation that is independent of the recruitment of Kin4. Thus, Spc72 provides a missing link between cytoplasmic MT function and components of the SPOC..
14. Clare L. Lawrence, Hiromi Maekawa, Jessica L. Worthington, Wolfgang Reiter, Caroline R.M. Wilkinson, Nic Jones, Regulation of Schizosaccharomyces pombe Atf1 protein levels by Sty1-mediated phosphorylation and heterodimerization with Pcr1, Journal of Biological Chemistry, 10.1074/jbc.M608526200, 282, 8, 5160-5170, 2007.02, The Atf1 transcription factor plays a vital role in the ability of Schizosaccharomyces pombe cells to respond to various stress conditions. It regulates the expression of many genes in a stress-dependent manner, and its function is dependent upon the stress-activated MAPK, Sty1/Spc1. Moreover, Atf1 is directly phosphorylated by Sty1. Here we have investigated the role of such phosphorylation. Atf1 protein accumulates following stress, and this accumulation is lost in a strain defective in the Sty1 signaling pathway. In addition, accumulation of a mutant Atf1 protein that can no longer be phosphorylated is lost. Measurement of the half-life of Atf1 demonstrates that changes in Atf1 stability are responsible for this accumulation. Atf1 stability is also regulated by its heterodimeric partner, Pcr1. Similarly, Pcr1 levels are regulated by Atf1. Thus multiple pathways exist that ensure that Atf1 levels are appropriately regulated. Phosphorylation of Atf1 is important for cells to mount a robust response to H2O2 stress, because the Atf1 phospho-mutant displays sensitivity to this stress, and induction of gene expression is lower than that observed in wild-type cells. Surprisingly, however, loss of Atf1 phosphorylation does not lead to the complete loss of stress-activated expression of Atf1 target genes. Accordingly, the Atf1 phospho-mutant does not display the same overall stress sensitivities as the atf1 deletion mutant. Taken together, these data suggest that Sty1 phosphorylation of Atf1 is not required for activation of Atf1 per se but rather for modulating its stability..
15. Yasuhiro Araki, Corine K. Lau, Hiromi Maekawa, Sue L. Jaspersen, Thomas H. Giddings, Elmar Schiebel, Mark Winey, The Saccharomyces cerevisiae spindle pole body (SPB) component Nbp1p is required for SPB membrane insertion and interacts with the integral membrane proteins Ndc1p and Mps2p, Molecular biology of the cell, 10.1091/mbc.E05-07-0668, 17, 4, 1959-1970, 2006.04, The spindle pole body (SPB) in Saccharomyces cerevisiae functions to nucleate and organize spindle microtubules, and it is embedded in the nuclear envelope throughout the yeast life cycle. However, the mechanism of membrane insertion of the SPB has not been elucidated. Ndc1p is an integral membrane protein that localizes to SPBs, and it is required for insertion of the SPB into the nuclear envelope during SPB duplication. To better understand the function of Ndc1p, we performed a dosage suppressor screen using the ndc1-39 temperature-sensitive allele. We identified an essential SPB component, Nbp1p. NBP1 shows genetic interactions with several SPB genes in addition to NDC1, and two-hybrid analysis revealed that Nbp1p binds to Ndc1p. Furthermore, Nbp1p is in the Mps2p-Bbp1p complex in the SPB. Immunoelectron microscopy confirmed that Nbp1p localizes to the SPB, suggesting a function at this location. Consistent with this hypothesis, nbp1-td (a degron allele) cells fail in SPB duplication upon depletion of Nbp1p. Importantly, these cells exhibit a "dead" SPB phenotype, similar to cells mutant in MPS2, NDC1, or BBP1. These results demonstrate that Nbp1p is a SPB component that acts in SPB duplication at the point of SPB insertion into the nuclear envelope..
16. Carsten Janke, Maria M. Magiera, Nicole Rathfelder, Christof Taxis, Simone Reber, Hiromi Maekawa, Alexandra Moreno-Borchart, Georg Doenges, Etienne Schwob, Elmar Schiebel, Michael Knop, A versatile toolbox for PCR-based tagging of yeast genes
New fluorescent proteins, more markers and promoter substitution cassettes, Yeast, 10.1002/yea.1142, 21, 11, 947-962, 2004.08, Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches..
17. Hiromi Maekawa, Elmar Schiebel, Cdk1-Clb4 controls the interaction of astral microtubule plus ends with subdomains of the daughter cell cortex, Genes and Development, 10.1101/gad.298704, 18, 14, 1709-1724, 2004.07, As in many polarized cells, spindle alignment in yeast is essential and cell cycle regulated. A key step that governs spindle alignment is the selective binding of the Kar9 protein to only one of the two spindle pole bodies (SPBs). It has been suggested that cyclin-dependent kinase Cdc28, in complex with cyclin Clb4, associates only with the SPB in the mother cell and so prevents Kar9 binding to this SPB. However, here we show that the nonoverexpressed Clb4 associates with the budward-directed SPB through Kar9. Cdc28-Clb4 then uses Kar9 as a carrier to move from this SPB to the plus ends of astral microtubules, where Cdc28-Clb4 regulates the interactions between microtubule ends and subdomains of the bud cortex. In the absence of Cdc28-Clb4 activity (G1/S phase), astral microtubules interact with the bud tip in a manner dependent on actin, Myo2, and Kar9. Coincidentally with reaching the bud cortex in S phase, Cdc28-Clb4 facilitates the dissociation of the microtubule bud tip interaction and their capture by the bud neck. This transition prevents the preanaphase spindle from becoming prematurely pulled into the bud. Thus, Cdc28-Clb4 facilitates spindle alignment by regulating the interaction of astral microtubules with subdomains of the bud cortex..
18. Hiromi Maekawa, Elmar Schiebel, CLIP-170 family members
A motor-driven ride to microtubule plus ends, Developmental Cell, 10.1016/j.devcel.2004.05.017, 6, 6, 746-748, 2004.01, CLIP-170 family proteins regulate microtubule plus end dynamics. Two reports published in this issue of Developmental Cell show that Bik1 and tip1p, the CLIP-170-like proteins of budding and fission yeast, are carried to microtubule plus ends by kinesin motor proteins. These findings indicate a complex interplay between microtubule-associated proteins and suggest a novel mechanism by which kinesin proteins stabilize microtubules..
19. Takeo Usui, Hiromi Maekawa, Gislene Pereira, Elmar Schiebel, The XMAP215 homologue Stu2 at yeast spindle pole bodies regulates microtubule dynamics and anchorage, EMBO Journal, 10.1093/emboj/cdg459, 22, 18, 4779-4793, 2003.09, The yeast protein Stu2 belongs to the XMAP215 family of conserved microtubule-binding proteins which regulate microtubule plus end dynamics. XMAP215-related proteins also bind to centrosomes and spindle pole bodies (SPBs) through proteins like the mammalian transforming acidic coiled coil protein TACC or the yeast Spc72. We show that yeast Spc72 has two distinct domains involved in microtubule organization. The essential 100 N-terminal amino acids of Spc72 interact directly with the γ-tubulin complex, and an adjacent non-essential domain of Spc72 mediates binding to Stu2. Through these domains, Spc72 brings Stu2 and the γ-tubulin complex together into a single complex. Manipulation of Spc72-Stu2 interaction at SPBs compromises the anchorage of astral microtubules at the SPB and surprisingly also influences the dynamics of microtubule plus ends. Permanently tethering Stu2 to SPBs by fusing it to a version of Spc72 that lacks the Stu2-binding site in part complements these defects in a manner which is dependent upon the microtubule-binding domain of Stu2. Thus, the SPB-associated Spc72-Stu2 complex plays a key role in regulating microtubule properties..
20. Hiromi Maekawa, Takeo Usui, Michael Knop, Elmar Schiebel, Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate bud cortex interactions, EMBO Journal, 10.1093/emboj/cdg063, 22, 3, 438-449, 2003.02, The budding yeast spindle aligns along the motherbud axis through interactions between cytoplasmic microtubules (CMs) and the cell cortex. Kar9, in complex with the EB1-related protein Bim1, mediates contacts of CMs with the cortex of the daughter cell, the bud. Here we established a novel series of events that target Kar9 to the bud cortex. First, Kar9 binds to spindle pole bodies (SPBs) in G1 of the cell cycle. Secondly, in G1/S the yeast Cdk1, Cdc28, associates with SPBs and phosphorylates Kar9. Thirdly, Kar9 and Cdc28 then move from the SPB to the plus end of CMs directed towards the bud. This movement is dependent upon the microtubule motor protein Kip2. Cdc28 activity is required to concentrate Kar9 at the plus end of CMs and hence to establish contacts with the bud cortex. The Cdc28-regulated localization of Kar9 is therefore an integral part of the program that aligns spindles..
21. Masayo Morishita, Fusako Morimoto, Kenji Kitamura, Takako Koga, Yasuhisa Fukui, Hiromi Maekawa, Ichiro Yamashita, Chikashi Shimoda, Phosphatidylinositol 3-phosphate 5-kinase is required for the cellular response to nutritional starvation and mating pheromone signals in Schizosaccharomyces pombe, Genes to Cells, 10.1046/j.1356-9597.2001.00510.x, 7, 2, 199-215, 2002.03, Background: Phosphatidylinositol (3,5) bisphosphate, which is converted from phosphatidylinositol 3-phosphate by phosphatidylinositol 3-phosphate 5-kinase, is implicated in vacuolar functions and the sorting of cell surface proteins within endosomes in the endocytic pathway of budding yeast. A homologous protein, SpFab1p, has been found in the fission yeast Schizosaccharomyces pombe, but its role is not known. Results: Here we report that SpFab1p is encoded by ste12+ known as a fertility gene in S. pombe. The ste12 mutant grew normally under stress-free conditions, but was highly vacuolated and swelled at high temperatures and under starvation conditions. In nitrogen-free medium, ste12 cells were arrested in G1 phase, but partially defective in the expression of genes responsible for mating and meiosis. The ste12 mutant was defective both in the production of, and in the response to, mating pheromones. The amount of the pheromone receptor protein Map3p, was substantially decreased in ste12 cells. Map3p was transported to the cell surface, then internalized and eventually transported to the vacuolar lumen, even in the ste12 mutant. Conclusion: The results indicate that phosphatidylinositol(3,5)bisphosphate is essential for cellular responses to various stresses and for the mating pheromone signalling under starvation conditions..
22. Kenji Kitamura, Hiromi Maekawa, Chikashi Shimoda, Fission yeast ste9, a homolog of Hct1/Cdh1 and fizzy-related, is a novel negative regulator of cell cycle progression during G1-phase, Molecular Biology of the Cell, 10.1091/mbc.9.5.1065, 9, 5, 1065-1080, 1998.01, When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G1-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G1-phase. The role of ste9+ in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G1-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G1/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10(ts), cells arrested in G1-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G1-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G1 progression possibly by controlling the amount of the mitotic cyclin in the G1-phase..
23. Hiromi Maekawa, Kenji Kitamura, Chikashi Shimoda, The Ste16 WD-repeat protein regulates cell cycle progression under starvation through the Rum1 protein in Schizosaccharomyces pombe, Current Genetics, 10.1007/s002940050305, 33, 1, 29-37, 1998.01, The haploid cells of the fission yeast, Schizosaccharomyces pombe, are arrested in the G1-phase by nitrogen starvation and are committed to sexual reproduction (mating and sporulation). We isolated the sterile mutants which were defective in G1 arrest following nitrogen starvation. Genetic analysis of these mutants defined a single locus designated as ste16. The nucleotide sequence revealed that ste16+ encodes an 82-kDa protein containing eight WD40-repeats in its carboxy terminal half. The ste16 disruptant was viable, but arrested the cell cycle in the G2-phase after the nutritional down-shift. When transferred to fresh growth medium, the G2-arrested ste16Δ haploids resumed the mitotic cycle from the S-phase, resulting in diploidization. This diploidization phenomenon was completely suppressed by the null mutation of rum1 encoding the inhibitor of Cdc2 kinase. As the Rum1 protein level was remarkably elevated in the ste16Δ, the Ste16 protein negatively controls the Rum1 level. The loss of function of ste16 disturbs the cell-cycle progression and impairs the mechanism for the maintenance of ploidy..
24. Hiromi Maekawa, Tomoko Nakagawa, Yoko Uno, Kenji Kitamura, Chikashi Shimoda, The ste13+ gene encoding a putative RNA helicase is essential for nitrogen starvation-induced G1 arrest and initiation of sexual development in the fission yeast Schizosaccharomyces pombe, Mgg Molecular & General Genetics, 10.1007/BF00583896, 244, 5, 456-464, 1994.09, When the fission yeast Schizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. The ste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear that ste13+ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone the ste13+ gene from an S. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts that ste13+ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished the ste13+ functions. The predicted Ste13 protein is 72% identical to the Drosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA in S. pombe suppresses the ste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development..