Biology subjects

Takegawa, K.

Publications and source records attributed to Takegawa, K..

5 recordsLinked to original sources

bioRxiv2025

Centromere chromatin may regulate the Flip-flop type of the Mating Type Switching in methylotrophic yeast Ogataea polymorpha

Centromere chromatin has been implicated in silencing the mating type (MAT) locus, which resides within a chromosomal region occupied by OpCse4, the CENP-A homolog, in the Flip-flop type of Mating Type Switching (MTS) in the methylotrophic yeast Ogataea polymorpha. In this study, we investigated the role of centromere chromatin in the recombination event between MAT-adjacent inverted repeats (IRs) that drives MTS. Our results demonstrate that the position of the centromere-proximal IR is critical for efficient recombination between the IRs. Mutants lacking functional OpCse4 or its chaperone OpScm3 exhibit MTS-deficient phenotypes, supporting an active role for centromere chromatin in facilitating MTS. Additionally, we identified OpRad6 and OpBre1 as essential for MTS, and found that deletion of the C-terminal acidic tail of OpRad6 alone is sufficient to disrupt switching. This suggests that monoubiquitylated histone H2B may contribute to MTS either directly or indirectly. Collectively, our findings highlight a direct and functional involvement of centromere chromatin in promoting Flip-flop type MTS in O. polymorpha.

molecular biology↗

bioRxiv2024

Structural basis for the strict substrate specificity of beta-D-galactofuranosidase from Streptomyces sp. JHA19

D-Galactofuranose (Galf) is widely distributed in polysaccharides and glycoconjugates of bacteria, filamentous fungi, and protozoa. The biosynthetic and degradation pathways of Galf in pathogens have attracted attention as potential targets for drug development. {beta}-D-Galactofuranosidase (Galf-ase) releases Galf from the non-reducing ends of glycans. Galf-ase activity is often exhibited by -L-arabinofuranosidases, which hydrolyze a similar substrate. Several Galf-specific Galf-ases that cleave only Galf and not L-arabinofuranose (Araf) have recently been identified in the glycoside hydrolase (GH) families 2, 5, and 43. However, the structural basis of how they discriminate the substrates is unknown. ORF1110, belonging to GH2, is the first identified Galf-specific Galf-ase isolated from Streptomyces sp. JHA19. Here, we solved the crystal structure of ORF1110 in complex with a mechanism-based potent inhibitor, D-iminogalactitol (Ki = 65 M). ORF1110 binds to the C5-C6 hydroxy groups of D-iminogalactitol with an extensive and integral hydrogen bond network. This result suggests that in the case of Araf, which lacks the C6 hydroxymethyl group, this network is not formed. The domain structure of ORF1110 is similar to that of {beta}-glucuronidases and {beta}-galactosidases, which belong to the same GH2 family and hydrolyze pyranose substrates. However, their active site structures were completely different. A predicted structure of the C-terminal Abf domain of ORF1110 was very similar to the carbohydrate-binding module family 42, which binds Araf, and pockets that may bind Galf were present.

biochemistry↗

bioRxiv2023

Polarity-dependent expression and localization of secretory glucoamylase mRNA in filamentous fungal cells

In multinuclear and multicellular filamentous fungi little is known about how mRNAs encoding secreted enzymes are transcribed and localized spatiotemporally. To better understand this process we visualized mRNA encoding GlaA, a glucoamylase secreted in large amounts by the industrial filamentous fungus Aspergillus oryzae, in living cells by the MS2 system. We found that glaA mRNA was significantly transcribed and localized near the hyphal tip and septum, which are the sites of protein secretion. We also revealed that glaA mRNA exhibits long-range dynamics in the vicinity of the endoplasmic reticulum (ER) in a manner that is dependent on the microtubule motor proteins kinesin-1 and kinesin-3, but independent of early endosomes. Moreover, we elucidated that the regulatory mechanisms of glaA mRNA by stress granules and processing bodies were different under high temperature and ER stress. Collectively, this study uncovers a dynamic regulatory mechanism of mRNA encoding a secretory enzyme in filamentous fungi.

cell biology↗

bioRxiv2021

SIN-like pathway kinases regulate the end of mitosis in the methylotrophic yeast Ogataea polymorpha

The Mitotic exit network (MEN) is a conserved signalling pathway essential for termination of mitosis in the budding yeast Saccharomyces cerevisiae. All MEN components are highly conserved in the methylotrophic budding yeast Ogataea polymorpha, except for Cdc15 kinase. Amongst O. polymorpha protein kinases that have some similarity to ScCdc15, only two had no other obvious homologues in S. cerevisiae and these were named OpHCD1 and OpHCD2 for homologue candidate of ScCdc15. A search in other yeast species revealed that OpHcd2 has an armadillo type fold in the C-terminal region as found in SpCdc7 kinases of the fission yeast Schizosaccharomyces pombe, which are homologues of ScCdc15; while OpHcd1 is homologous to SpSid1 kinase, a component of the Septation Initiation Network (SIN) of S. pombe not present in the MEN. Since the deletion of either OpHCD1 or OpHCD2 resulted in lethality under standard growth conditions, conditional mutants were constructed by introducing an ATP analog sensitive mutation. For OpHCD2, we constructed and used new genetic tools for O. polymorpha that combined the Tet promoter and the improved auxin-degron systems. Conditional mutants for OpHCD1 and OpHCD2 exhibited significant delay in late anaphase and defective cell separation, suggesting that both genes have roles in mitotic exit and cytokinesis. These results suggest a SIN-like signalling pathway regulates termination of mitosis in O. polymorpha and that the loss of Sid1/Hcd1 kinase in the MEN occurred relatively recently during the evolution of budding yeast.

molecular biology↗

bioRxiv2019

Biosynthesis of β-(1->5)-Galactofuranosyl Chains of Fungal-Type and O-Mannose-Type Galactomannans within the Invasive Pathogen Aspergillus fumigatus

The pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise -(1[->]2)-/-(1[->]6)-mannan and {beta}-(1[->]5)-/{beta}-(1[->]6)-galactofuranosyl chains. We previously revealed that GfsA is a {beta}-galactofuranoside {beta}-(1[->]5)-galactofuranosyltransferase involved in the biosynthesis of {beta}-(1[->]5)-galactofuranosyl chains. Here, we clarified the entire biosynthesis of {beta}-(1[->]5)-galactofuranosyl chains in A. fumigatgus. Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are {beta}-galactofuranoside {beta}-(1[->]5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize {beta}-(1[->]5)-galactofuranosyl oligomers up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from the strains {Delta}gfsB, {Delta}gfsC, {Delta}gfsAC, and {Delta}gfsABC revealed that GfsA and GfsC synthesized all {beta}-(1[->]5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans, and GfsB exhibited limited function in A. fumigatus. The loss of {beta}-(1[->]5)-galactofuranosyl residues decreased the hyphal growth rate and conidia formation ability as well as increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immune-compromised mice.\n\nIMPORTANCE{beta}-(1[->]5)-galactofuranosyl residues are widely distributed in the subphylum Pezisomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of {beta}-(1[->]5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for their understanding. In this study, we show that GfsA and GfsC are responsible for the biosynthesis of all {beta}-(1[->]5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicates that {beta}-(1[->]5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of {beta}-(1[->]5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the subphylum Pezisomycotina.

microbiology↗