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Gregory Jedd - One of the best experts on this subject based on the ideXlab platform.
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Multiple modes for gatekeeping at fungal cell-to-cell channels.
Molecular microbiology, 2012Co-Authors: Gregory Jedd, Laurent PieuchotAbstract:Cell-to-cell channels appear to be indispensable for successful multicellular organization and arose independently in animals, plants and fungi. Most of the fungi obtain nutrients from the environment by growing in an exploratory and invasive manner, and this ability depends on multicellular filaments known as hyphae. These cells grow by tip extension and can be divided into compartments by cell walls that typically retain a central pore that allows intercellular transport and cooperation. In the major clade of filamentous Ascomycota, integrity of this coenocytic organization is maintained by Woronin Body organelles, which function as emergency patches of septal pores. In this issue of Molecular Microbiology, Bleichrodt and co-workers show that Woronin bodies can also form tight reversible associations with the pore and further link this to variation in levels of compartmental gene expression. These data define an additional modality of Woronin Body-dependent gatekeeping. This commentary focuses on the implications of this work and the potential role of different modes of pore gating in controlling the growth and development of fungal tissues.
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Multiple modes for gatekeeping at fungal cell‐to‐cell channels
Molecular Microbiology, 2012Co-Authors: Gregory Jedd, Laurent PieuchotAbstract:Summary Cell-to-cell channels appear to be indispensable for successful multicellular organization and arose independently in animals, plants and fungi. Most of the fungi obtain nutrients from the environment by growing in an exploratory and invasive manner, and this ability depends on multicellular filaments known as hyphae. These cells grow by tip extension and can be divided into compartments by cell walls that typically retain a central pore that allows intercellular transport and cooperation. In the major clade of filamentous Ascomycota, integrity of this coenocytic organization is maintained by Woronin Body organelles, which function as emergency patches of septal pores. In this issue of Molecular Microbiology, Bleichrodt and co-workers show that Woronin bodies can also form tight reversible associations with the pore and further link this to variation in levels of compartmental gene expression. These data define an additional modality of Woronin Body-dependent gatekeeping. This commentary focuses on the implications of this work and the potential role of different modes of pore gating in controlling the growth and development of fungal tissues.
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intrinsically disordered proteins aggregate at fungal cell to cell channels and regulate intercellular connectivity
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Monika Tjota, Vignesh Raman, Karthik Balakrishna Chandrababu, Daiwen Yang, Limsoon Wong, Laurent Pieuchot, Gregory JeddAbstract:Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin Body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin Body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity.
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Fungal evo-devo: organelles and multicellular complexity.
Trends in cell biology, 2010Co-Authors: Gregory JeddAbstract:Peroxisome-derived Woronin bodies of the Ascomycota phyla, and the endoplasmic reticulum (ER)-derived septal pore cap (SPC) of the Basidiomycota, are both fungal organelles that prevent cytoplasmic bleeding when multicellular hyphal filaments are wounded. Analysis of Woronin Body constituent proteins suggests that these organelles evolved in part through gene duplication and co-opting of non-essential genes for new functions, indicating that new organelles can arise through typical evolutionary mechanisms. Interestingly, clades possessing the Woronin Body and SPC also produce the largest and most complex multicellular fungal reproductive structures. Certain Woronin Body and SPC mutants have defects in growth and development, suggesting functions beyond cellular wound healing. I argue that studying these specialized systems will help to reveal the basis for fungal diversity and provide general principles for co-evolution of organelles and multicellular complexity.
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A tether for Woronin Body inheritance is associated with evolutionary variation in organelle positioning.
PLoS genetics, 2009Co-Authors: Fangfang Liu, Wilson Low, Julian Lai, Gregory JeddAbstract:Eukaryotic organelles evolve to support the lifestyle of evolutionarily related organisms. In the fungi, filamentous Ascomycetes possess dense-core organelles called Woronin bodies (WBs). These organelles originate from peroxisomes and perform an adaptive function to seal septal pores in response to cellular wounding. Here, we identify Leashin, an organellar tether required for WB inheritance, and associate it with evolutionary variation in the subcellular pattern of WB distribution. In Neurospora, the leashin (lah) locus encodes two related adjacent genes. N-terminal sequences of LAH-1 bind WBs via the WB–specific membrane protein WSC, and C-terminal sequences are required for WB inheritance by cell cortex association. LAH-2 is localized to the hyphal apex and septal pore rim and plays a role in colonial growth. In most species, WBs are tethered directly to the pore rim, however, Neurospora and relatives have evolved a delocalized pattern of cortex association. Using a new method for the construction of chromosomally encoded fusion proteins, marker fusion tagging (MFT), we show that a LAH-1/LAH-2 fusion can reproduce the ancestral pattern in Neurospora. Our results identify the link between the WB and cell cortex and suggest that splitting of leashin played a key role in the adaptive evolution of organelle localization.
Katsuhiko Kitamoto - One of the best experts on this subject based on the ideXlab platform.
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A Large Nonconserved Region of the Tethering Protein Leashin Is Involved in Regulating the Position, Movement, and Function of Woronin Bodies in Aspergillus oryzae
2016Co-Authors: Pei Han, Feng Jie Jin, Jun-ichi Maruyama, Katsuhiko KitamotoAbstract:TheWoronin Body is a Pezizomycotina-specific organelle that is typically tethered to the septum, but upon hyphal wounding, it plugs the septal pore to prevent excessive cytoplasmic loss. Leashin (LAH) is a largeWoronin Body tethering protein that con-tains highly conserved N- and C-terminal regions and a long (2,500-amino-acid) nonconserved middle region. As the involve-ment of the nonconserved region inWoronin Body function has not been investigated, here, we functionally characterized indi-vidual regions of the LAH protein of Aspergillus oryzae (AoLAH). In an Aolah disruptant, noWoronin bodies were tethered to the septum, and hyphae had a reduced ability to prevent excessive cytoplasmic loss upon hyphal wounding. Localization analysis revealed that the N-terminal region of AoLAH associated withWoronin bodies dependently on AoWSC, which is homologous to Neurospora crassaWSC (Woronin Body sorting complex), and that the C-terminal region was localized to the septum. Elastic movement ofWoronin bodies was observed when visualized with an AoLAHN-terminal-region–enhanced green fluorescent protein (EGFP) fusion protein. An N- and C-terminal fusion construct lacking the nonconserved middle region of AoLAHwas sufficient for the tethering ofWoronin bodies to the septum. However, Woronin bodies were located closer to the septum and exhibited impaired elastic movement. Moreover, expression of middle-region-deleted AoLAH in the Aolah disruptant did not restore the ability to prevent excessive cytoplasmic loss. These findings indicate that the nonconserved middle region of AoLAH has functional importance for regulating the position, movement, and function ofWoronin bodies. Filamentous fungi grow via a polarized tip extension, whichforms tubular filaments called hyphae that are further divide
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cell biology of the koji mold aspergillus oryzae
Bioscience Biotechnology and Biochemistry, 2015Co-Authors: Katsuhiko KitamotoAbstract:Koji mold, Aspergillus oryzae, has been used for the production of sake, miso, and soy sauce for more than one thousand years in Japan. Due to the importance, A. oryzae has been designated as the national micro-organism of Japan (Koku-kin). A. oryzae has been intensively studied in the past century, with most investigations focusing on breeding techniques and developing methods for Koji making for sake brewing. However, the understanding of fundamental biology of A. oryzae remains relatively limited compared with the yeast Saccharomyces cerevisiae. Therefore, we have focused on studying the cell biology including live cell imaging of organelles, protein vesicular trafficking, autophagy, and Woronin Body functions using the available genomic information. In this review, I describe essential findings of cell biology of A. oryzae obtained in our study for a quarter of century. Understanding of the basic biology will be critical for not its biotechnological application, but also for an understanding of the fundamental biology of other filamentous fungi.
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A large nonconserved region of the tethering protein Leashin is involved in regulating the position, movement, and function of Woronin bodies in Aspergillus oryzae.
Eukaryotic cell, 2014Co-Authors: Pei Han, Feng Jie Jin, Jun-ichi Maruyama, Katsuhiko KitamotoAbstract:ABSTRACT The Woronin Body is a Pezizomycotina-specific organelle that is typically tethered to the septum, but upon hyphal wounding, it plugs the septal pore to prevent excessive cytoplasmic loss. Leashin (LAH) is a large Woronin Body tethering protein that contains highly conserved N- and C-terminal regions and a long (∼2,500-amino-acid) nonconserved middle region. As the involvement of the nonconserved region in Woronin Body function has not been investigated, here, we functionally characterized individual regions of the LAH protein of Aspergillus oryzae (AoLAH). In an Aolah disruptant, no Woronin bodies were tethered to the septum, and hyphae had a reduced ability to prevent excessive cytoplasmic loss upon hyphal wounding. Localization analysis revealed that the N-terminal region of AoLAH associated with Woronin bodies dependently on AoWSC, which is homologous to Neurospora crassa WSC (Woronin Body sorting complex), and that the C-terminal region was localized to the septum. Elastic movement of Woronin bodies was observed when visualized with an AoLAH N-terminal-region–enhanced green fluorescent protein (EGFP) fusion protein. An N- and C-terminal fusion construct lacking the nonconserved middle region of AoLAH was sufficient for the tethering of Woronin bodies to the septum. However, Woronin bodies were located closer to the septum and exhibited impaired elastic movement. Moreover, expression of middle-region-deleted AoLAH in the Aolah disruptant did not restore the ability to prevent excessive cytoplasmic loss. These findings indicate that the nonconserved middle region of AoLAH has functional importance for regulating the position, movement, and function of Woronin bodies.
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Expanding functional repertoires of fungal peroxisomes: contribution to growth and survival processes
Frontiers in physiology, 2013Co-Authors: Jun-ichi Maruyama, Katsuhiko KitamotoAbstract:It has long been regarded that the primary function of fungal peroxisomes is limited to the β-oxidation of fatty acids, as mutants lacking peroxisomal function fail to grow in minimal medium containing fatty acids as the sole carbon source. However, studies in filamentous fungi have revealed that peroxisomes have diverse functional repertoires. This review describes the essential roles of peroxisomes in the growth and survival processes of filamentous fungi. One such survival mechanism involves the Woronin Body, a Pezizomycotina-specific organelle that plugs the septal pore upon hyphal lysis to prevent excessive cytoplasmic loss. A number of reports have demonstrated that Woronin bodies are derived from peroxisomes. Specifically, the Woronin Body protein Hex1 is targeted to peroxisomes by peroxisomal targeting sequence 1 (PTS1) and forms a self-assembled structure that buds from peroxisomes to form the Woronin Body. Peroxisomal deficiency reduces the ability of filamentous fungi to prevent excessive cytoplasmic loss upon hyphal lysis, indicating that peroxisomes contribute to the survival of these multicellular organisms. Peroxisomes were also recently found to play a vital role in the biosynthesis of biotin, which is an essential cofactor for various carboxylation and decarboxylation reactions. In biotin-prototrophic fungi, peroxisome-deficient mutants exhibit growth defects when grown on glucose as a carbon source due to biotin auxotrophy. The biotin biosynthetic enzyme BioF (7-keto-8-aminopelargonic acid synthase) contains a PTS1 motif that is required for both peroxisomal targeting and biotin biosynthesis. In plants, the BioF protein contains a conserved PTS1 motif and is also localized in peroxisomes. These findings indicate that the involvement of peroxisomes in biotin biosynthesis is evolutionarily conserved between fungi and plants, and that peroxisomes play a key role in fungal growth.
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Hyphal heterogeneity in Aspergillus oryzae is the result of dynamic closure of septa by Woronin bodies.
Molecular microbiology, 2012Co-Authors: Robertjan Bleichrodt, Katsuhiko Kitamoto, Jun-ichi Maruyama, G. Jerre Van Veluw, Brand Recter, Han A B WostenAbstract:Summary Hyphae of higher fungi are compartmentalized by septa. These septa contain a central pore that allows for inter-compartmental and inter-hyphal cytoplasmic streaming. The cytoplasm within the mycelium is therefore considered to be a continuous system. In this study, however, we demonstrate by laser dissection that 40% of the apical septa of exploring hyphae of Aspergillus oryzae are closed. Closure of septa correlated with the presence of a peroxisome-derived organelle, known as Woronin Body, near the septal pore. The location of Woronin bodies in the hyphae was dynamic and, as a result, plugging of the septal pore was reversible. Septal plugging was abolished in a ΔAohex1 strain that cannot form Woronin bodies. Notably, hyphal heterogeneity was also affected in the ΔAohex1 strain. Wild-type strains of A. oryzae showed heterogeneous distribution of GFP between neighbouring hyphae at the outer part of the colony when the reporter was expressed from the promoter of the glucoamylase gene glaA or the α-glucuronidase gene aguA. In contrast, GFP fluorescence showed a normal distribution in the case of the ΔAohex1 strain. Taken together, it is concluded that Woronin bodies maintain hyphal heterogeneity in a fungal mycelium by impeding cytoplasmic continuity.
Jun-ichi Maruyama - One of the best experts on this subject based on the ideXlab platform.
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A Large Nonconserved Region of the Tethering Protein Leashin Is Involved in Regulating the Position, Movement, and Function of Woronin Bodies in Aspergillus oryzae
2016Co-Authors: Pei Han, Feng Jie Jin, Jun-ichi Maruyama, Katsuhiko KitamotoAbstract:TheWoronin Body is a Pezizomycotina-specific organelle that is typically tethered to the septum, but upon hyphal wounding, it plugs the septal pore to prevent excessive cytoplasmic loss. Leashin (LAH) is a largeWoronin Body tethering protein that con-tains highly conserved N- and C-terminal regions and a long (2,500-amino-acid) nonconserved middle region. As the involve-ment of the nonconserved region inWoronin Body function has not been investigated, here, we functionally characterized indi-vidual regions of the LAH protein of Aspergillus oryzae (AoLAH). In an Aolah disruptant, noWoronin bodies were tethered to the septum, and hyphae had a reduced ability to prevent excessive cytoplasmic loss upon hyphal wounding. Localization analysis revealed that the N-terminal region of AoLAH associated withWoronin bodies dependently on AoWSC, which is homologous to Neurospora crassaWSC (Woronin Body sorting complex), and that the C-terminal region was localized to the septum. Elastic movement ofWoronin bodies was observed when visualized with an AoLAHN-terminal-region–enhanced green fluorescent protein (EGFP) fusion protein. An N- and C-terminal fusion construct lacking the nonconserved middle region of AoLAHwas sufficient for the tethering ofWoronin bodies to the septum. However, Woronin bodies were located closer to the septum and exhibited impaired elastic movement. Moreover, expression of middle-region-deleted AoLAH in the Aolah disruptant did not restore the ability to prevent excessive cytoplasmic loss. These findings indicate that the nonconserved middle region of AoLAH has functional importance for regulating the position, movement, and function ofWoronin bodies. Filamentous fungi grow via a polarized tip extension, whichforms tubular filaments called hyphae that are further divide
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A large nonconserved region of the tethering protein Leashin is involved in regulating the position, movement, and function of Woronin bodies in Aspergillus oryzae.
Eukaryotic cell, 2014Co-Authors: Pei Han, Feng Jie Jin, Jun-ichi Maruyama, Katsuhiko KitamotoAbstract:ABSTRACT The Woronin Body is a Pezizomycotina-specific organelle that is typically tethered to the septum, but upon hyphal wounding, it plugs the septal pore to prevent excessive cytoplasmic loss. Leashin (LAH) is a large Woronin Body tethering protein that contains highly conserved N- and C-terminal regions and a long (∼2,500-amino-acid) nonconserved middle region. As the involvement of the nonconserved region in Woronin Body function has not been investigated, here, we functionally characterized individual regions of the LAH protein of Aspergillus oryzae (AoLAH). In an Aolah disruptant, no Woronin bodies were tethered to the septum, and hyphae had a reduced ability to prevent excessive cytoplasmic loss upon hyphal wounding. Localization analysis revealed that the N-terminal region of AoLAH associated with Woronin bodies dependently on AoWSC, which is homologous to Neurospora crassa WSC (Woronin Body sorting complex), and that the C-terminal region was localized to the septum. Elastic movement of Woronin bodies was observed when visualized with an AoLAH N-terminal-region–enhanced green fluorescent protein (EGFP) fusion protein. An N- and C-terminal fusion construct lacking the nonconserved middle region of AoLAH was sufficient for the tethering of Woronin bodies to the septum. However, Woronin bodies were located closer to the septum and exhibited impaired elastic movement. Moreover, expression of middle-region-deleted AoLAH in the Aolah disruptant did not restore the ability to prevent excessive cytoplasmic loss. These findings indicate that the nonconserved middle region of AoLAH has functional importance for regulating the position, movement, and function of Woronin bodies.
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Expanding functional repertoires of fungal peroxisomes: contribution to growth and survival processes
Frontiers in physiology, 2013Co-Authors: Jun-ichi Maruyama, Katsuhiko KitamotoAbstract:It has long been regarded that the primary function of fungal peroxisomes is limited to the β-oxidation of fatty acids, as mutants lacking peroxisomal function fail to grow in minimal medium containing fatty acids as the sole carbon source. However, studies in filamentous fungi have revealed that peroxisomes have diverse functional repertoires. This review describes the essential roles of peroxisomes in the growth and survival processes of filamentous fungi. One such survival mechanism involves the Woronin Body, a Pezizomycotina-specific organelle that plugs the septal pore upon hyphal lysis to prevent excessive cytoplasmic loss. A number of reports have demonstrated that Woronin bodies are derived from peroxisomes. Specifically, the Woronin Body protein Hex1 is targeted to peroxisomes by peroxisomal targeting sequence 1 (PTS1) and forms a self-assembled structure that buds from peroxisomes to form the Woronin Body. Peroxisomal deficiency reduces the ability of filamentous fungi to prevent excessive cytoplasmic loss upon hyphal lysis, indicating that peroxisomes contribute to the survival of these multicellular organisms. Peroxisomes were also recently found to play a vital role in the biosynthesis of biotin, which is an essential cofactor for various carboxylation and decarboxylation reactions. In biotin-prototrophic fungi, peroxisome-deficient mutants exhibit growth defects when grown on glucose as a carbon source due to biotin auxotrophy. The biotin biosynthetic enzyme BioF (7-keto-8-aminopelargonic acid synthase) contains a PTS1 motif that is required for both peroxisomal targeting and biotin biosynthesis. In plants, the BioF protein contains a conserved PTS1 motif and is also localized in peroxisomes. These findings indicate that the involvement of peroxisomes in biotin biosynthesis is evolutionarily conserved between fungi and plants, and that peroxisomes play a key role in fungal growth.
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Hyphal heterogeneity in Aspergillus oryzae is the result of dynamic closure of septa by Woronin bodies.
Molecular microbiology, 2012Co-Authors: Robertjan Bleichrodt, Katsuhiko Kitamoto, Jun-ichi Maruyama, G. Jerre Van Veluw, Brand Recter, Han A B WostenAbstract:Summary Hyphae of higher fungi are compartmentalized by septa. These septa contain a central pore that allows for inter-compartmental and inter-hyphal cytoplasmic streaming. The cytoplasm within the mycelium is therefore considered to be a continuous system. In this study, however, we demonstrate by laser dissection that 40% of the apical septa of exploring hyphae of Aspergillus oryzae are closed. Closure of septa correlated with the presence of a peroxisome-derived organelle, known as Woronin Body, near the septal pore. The location of Woronin bodies in the hyphae was dynamic and, as a result, plugging of the septal pore was reversible. Septal plugging was abolished in a ΔAohex1 strain that cannot form Woronin bodies. Notably, hyphal heterogeneity was also affected in the ΔAohex1 strain. Wild-type strains of A. oryzae showed heterogeneous distribution of GFP between neighbouring hyphae at the outer part of the colony when the reporter was expressed from the promoter of the glucoamylase gene glaA or the α-glucuronidase gene aguA. In contrast, GFP fluorescence showed a normal distribution in the case of the ΔAohex1 strain. Taken together, it is concluded that Woronin bodies maintain hyphal heterogeneity in a fungal mycelium by impeding cytoplasmic continuity.
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aoso protein accumulates at the septal pore in response to various stresses in the filamentous fungus aspergillus oryzae
Biochemical and Biophysical Research Communications, 2010Co-Authors: Jun-ichi Maruyama, Cristopher Salazar Escano, Katsuhiko KitamotoAbstract:Filamentous ascomycetes form hyphal networks that are compartmentalized by septa which have a perforated pore allowing the passage of cytoplasm and organelles between adjacent hyphal compartments. Thus, the septal pore may play an important role in the organized growth of multicellular organisms. Upon hyphal injury, the septal pore is plugged by a wound-healing organelle, known as the Woronin Body, to prevent excessive cytoplasmic leakage. However, the movement of proteins towards the septal pore in response to stress has not been extensively studied in filamentous fungi. In this study, we identified an Aspergillus oryzae protein, AoSO, which is homologous to the Neurospora crassa SO protein that was reported to accumulate at the septal pore in aging hyphae. The ΔAoso strain showed excessive cytoplasmic leakage upon hyphal injury similar to the Woronin Body-deficient strain ΔAohex1. Cellular localization studies using EGFP showed that AoSO accumulated at the septal pore adjacent to the injured compartment, while it was dispersed throughout the cytoplasm under normal growth conditions. These results indicate that AoSO plays a role in preventing excessive cytoplasmic leakage upon hyphal injury by accumulating at the septal pore. Furthermore, AoSO accumulated at the septal pore in response to various stresses, including low and high temperature, extreme acidic and alkaline pH, and nitrogen and carbon depletion. Physical stress induced by pulse laser treatment on a hyphal region at a distance from the septum caused accumulation of the AoSO protein at the septal pore within only a few minutes. This study presents a novel behavior in which a filamentous fungal protein relocalizes to the septal pore in response to various stresses.
Ulrich Kuck - One of the best experts on this subject based on the ideXlab platform.
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the ww domain protein pro40 is required for fungal fertility and associates with Woronin bodies
Eukaryotic Cell, 2007Co-Authors: Ines Engh, Christian Würtz, Hanspeter Rottensteiner, Konstanze Witzelschlomp, Hai Yu Zhang, Birgit Hoff, Minou Nowrousian, Ulrich KuckAbstract:Fruiting Body formation in ascomycetes is a highly complex process that is under polygenic control and is a fundamental part of the fungal sexual life cycle. However, the molecular determinants regulating this cellular process are largely unknown. Here we show that the sterile pro40 mutant is defective in a 120-kDa WW domain protein that plays a pivotal role in fruiting Body maturation of the homothallic ascomycete Sordaria macrospora. Although WW domains occur in many eukaryotic proteins, homologs of PRO40 are present only in filamentous ascomycetes. Complementation analysis with different pro40 mutant strains, using full-sized or truncated versions of the wild-type pro40 gene, revealed that the C terminus of PRO40 is crucial for restoring the fertile phenotype. Using differential centrifugation and protease protection assays, we determined that a PRO40-FLAG fusion protein is located within organelles. Further microscopic investigations of fusion proteins with DsRed or green fluorescent protein polypeptides showed a colocalization of PRO40 with HEX-1, a Woronin Body-specific protein. However, the integrity of Woronin bodies is not affected in mutant strains of S. macrospora and Neurospora crassa, as shown by fluorescence microscopy, sedimentation, and immunoblot analyses. We discuss the function of PRO40 in fruiting Body formation.
Hanspeter Rottensteiner - One of the best experts on this subject based on the ideXlab platform.
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Dynamin‐like protein‐dependent formation of Woronin bodies in Saccharomyces cerevisiae upon heterologous expression of a single protein
The FEBS journal, 2008Co-Authors: Christian Würtz, Wolfgang Schliebs, Ralf Erdmann, Hanspeter RottensteinerAbstract:Filamentous ascomycetes harbor Woronin bodies and glyoxysomes, two types of microbodies, within one cell at the same time. The dominant protein of the Neurospora crassa Woronin Body, HEX1, forms a hexagonal core crystal via oligomerization and evidence has accumulated that Woronin bodies bud off from glyoxysomes. We analyzed whether HEX1 is sufficient to induce Woronin Body formation upon heterologous expression in Saccharomyces cerevisiae, an organism devoid of this specialized organelle. In wild-type strain BY4742, initial import of HEX1 into existing peroxisomes enabled the formation of organelles with a hexagonal crystal. The observed structures mimicked the shape of genuine Woronin bodies, but exhibited a lower density and were significantly larger. Double-immunofluorescence analysis revealed that hexagonal HEX1 structures only occasionally co-localized with peroxisomal marker proteins, indicating that the Woronin-Body-like structures are well separated from peroxisomes. In cells lacking Vps1p and Dnm1p, dynamin-like proteins required for the division of peroxisomes, the Woronin-Body-like organelles remained attached to peroxisomes. The data indicate that Woronin bodies emerge after the formation of a HEX1 core crystal within peroxisomes followed by Vps1p- and Dnm1p-mediated fission.
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the ww domain protein pro40 is required for fungal fertility and associates with Woronin bodies
Eukaryotic Cell, 2007Co-Authors: Ines Engh, Christian Würtz, Hanspeter Rottensteiner, Konstanze Witzelschlomp, Hai Yu Zhang, Birgit Hoff, Minou Nowrousian, Ulrich KuckAbstract:Fruiting Body formation in ascomycetes is a highly complex process that is under polygenic control and is a fundamental part of the fungal sexual life cycle. However, the molecular determinants regulating this cellular process are largely unknown. Here we show that the sterile pro40 mutant is defective in a 120-kDa WW domain protein that plays a pivotal role in fruiting Body maturation of the homothallic ascomycete Sordaria macrospora. Although WW domains occur in many eukaryotic proteins, homologs of PRO40 are present only in filamentous ascomycetes. Complementation analysis with different pro40 mutant strains, using full-sized or truncated versions of the wild-type pro40 gene, revealed that the C terminus of PRO40 is crucial for restoring the fertile phenotype. Using differential centrifugation and protease protection assays, we determined that a PRO40-FLAG fusion protein is located within organelles. Further microscopic investigations of fusion proteins with DsRed or green fluorescent protein polypeptides showed a colocalization of PRO40 with HEX-1, a Woronin Body-specific protein. However, the integrity of Woronin bodies is not affected in mutant strains of S. macrospora and Neurospora crassa, as shown by fluorescence microscopy, sedimentation, and immunoblot analyses. We discuss the function of PRO40 in fruiting Body formation.
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The peroxin PEX14 of Neurospora crassa is essential for the biogenesis of both glyoxysomes and Woronin bodies
Traffic, 2007Co-Authors: David Managadze, Christian Würtz, Martin Sichting, Gerd Niehaus, Marten Veenhuis, Hanspeter RottensteinerAbstract:In the filamentous fungus Neurospora crassa, glyoxysomes and Woronin bodies coexist in the same cell. Because several glyoxysomal matrix proteins and also HEX1, the dominant protein of Woronin bodies, possess typical peroxisomal targeting signals, the question arises as to how protein targeting to these distinct yet related types of microbodies is achieved. Here we analyzed the function of the Neurospora ortholog of PEX14, an essential component of the peroxisomal import machinery. PEX14 interacted with both targeting signal receptors and was localized to glyoxysomes but was virtually absent from Woronin bodies. Nonetheless, a pex14 Delta mutant not only failed to grow on fatty acids because of a defect in glyoxysomal beta-oxidation but also suffered from cytoplasmic bleeding, indicative of a defect in Woronin Body-dependent septal pore plugging. Inspection of pex14 Delta mutant hyphae by fluorescence and electron microscopy indeed revealed the absence of Woronin bodies. When these cells were subjected to subcellular fractionation, HEX1 was completely mislocalized to the cytosol. Expression of GFP-HEX1 in wild-type mycelia caused the staining of Woronin bodies and also of glyoxysomes in a targeting signal-dependent manner. Our data support the view that Woronin bodies emerge from glyoxysomes through import of HEX1 and subsequent fission.
