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Elmar Schiebel - One of the best experts on this subject based on the ideXlab platform.
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evidence that the ipl1 sli15 aurora kinase incenp complex promotes chromosome bi orientation by altering kinetochore Spindle Pole connections
Cell, 2002Co-Authors: Gislene Pereira, Elmar Schiebel, Tomoyuki U Tanaka, Najma Rachidi, Carsten Janke, Marta Galova, Michael J R Stark, Kim NasmythAbstract:How sister kinetochores attach to microtubules from opposite Spindle Poles during mitosis (bi-orientation) remains poorly understood. In yeast, the ortholog of the Aurora B-INCENP protein kinase complex (Ipl1-Sli15) may have a role in this crucial process, because it is necessary to prevent attachment of sister kinetochores to microtubules from the same Spindle Pole. We investigated IPL1 function in cells that cannot replicate their chromosomes but nevertheless duplicate their Spindle Pole bodies (SPBs). Kinetochores detach from old SPBs and reattach to old and new SPBs with equal frequency in IPL1+ cells, but remain attached to old SPBs in ipl1 mutants. This raises the possibility that Ipl1-Sli15 facilitates bi-orientation by promoting turnover of kinetochore-SPB connections until traction of sister kinetochores toward opposite Spindle Poles creates tension in the surrounding chromatin.
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the role of the yeast Spindle Pole body and the mammalian centrosome in regulating late mitotic events
Current Opinion in Cell Biology, 2001Co-Authors: Gislene Pereira, Elmar SchiebelAbstract:Centrosomes of vertebrate cells and Spindle Pole bodies (SPBs) of fungi were first recognized through their ability to organize microtubules. Recent studies suggest that centrosomes and SPBs also have a function in the regulation of cell cycle progression, in particular in controlling late mitotic events. Regulators of mitotic exit and cytokinesis are associated with the SPB of budding and fission yeast. Elucidation of the molecular roles played by these regulators is helping to clarify the function of the SPB in controlling progression though mitosis.
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spc98p and spc97p of the yeast γ tubulin complex mediate binding to the Spindle Pole body via their interaction with spc110p
The EMBO Journal, 1997Co-Authors: Michael Knop, Elmar SchiebelAbstract:Previously, we have shown that the yeast γ-tubulin, Tub4p, forms a 6S complex with the Spindle Pole body components Spc98p and Spc97p. In this paper we report the purification of the Tub4p complex. It contained one molecule of Spc98p and Spc97p, and two or more molecules of Tub4p, but no other protein. We addressed how the Tub4p complex binds to the yeast microtubule organizing center, the Spindle Pole body (SPB). Genetic and biochemical data indicate that Spc98p and Spc97p of the Tub4p complex bind to the N-terminal domain of the SPB component Spc110p. Finally, we isolated a complex containing Spc110p, Spc42p, calmodulin and a 35 kDa protein, suggesting that these four proteins interact in the SPB. We discuss in a model, how the N-terminus of Spc110p anchors the Tub4p complex to the SPB and how Spc110p itself is embedded in the SPB.
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the Spindle Pole body component spc97p interacts with the γ tubulin of saccharomyces cerevisiae and functions in microtubule organization and Spindle Pole body duplication
The EMBO Journal, 1997Co-Authors: Michael Knop, Gislene Pereira, Katrin Grein, Silke Geissler, Elmar SchiebelAbstract:Previously, we have shown that the γ-tubulin Tub4p and the Spindle Pole body component Spc98p are involved in microtubule organization by the yeast microtubule organizing centre, the Spindle Pole body (SPB). In this paper we report the identification of SPC97 encoding an essential SPB component that is in association with the SPB substructures that organize the cytoplasmic and nuclear microtubules. Evidence is provided for a physical and functional interaction between Tub4p, Spc98p and Spc97p: first, temperature-sensitive spc97(ts) mutants are suppressed by high gene dosage of SPC98 or TUB4. Second, Spc97p interacts with Spc98p and Tub4p in the two-hybrid system. Finally, immunoprecipitation and fractionation studies revealed complexes containing Tub4p, Spc98p and Spc97p. Further support for a direct interaction of Tub4p, Spc98p and Spc97p comes from the toxicity of strong SPC97 overexpression which is suppressed by co-overexpression of TUB4 or SPC98. Analysis of temperature-sensitive spc97(ts) alleles revealed multiple Spindle defects. While spc97-14 cells are either impaired in SPB separation or mitotic Spindle formation, spc97-20 cells show an additional defect in SPB duplication. We discuss a model in which the Tub4p–Spc98p–Spc97p complex is part of the microtubule attachment site at the SPB.
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The Spindle Pole body component Spc97p interacts with the γ‐tubulin of Saccharomyces cerevisiae and functions in microtubule organization and Spindle Pole body duplication
The EMBO Journal, 1997Co-Authors: Michael Knop, Gislene Pereira, Katrin Grein, Silke Geissler, Elmar SchiebelAbstract:Previously, we have shown that the gamma-tubulin Tub4p and the Spindle Pole body component Spc98p are involved in microtubule organization by the yeast microtubule organizing centre, the Spindle Pole body (SPB). In this paper we report the identification of SPC97 encoding an essential SPB component that is in association with the SPB substructures that organize the cytoplasmic and nuclear microtubules. Evidence is provided for a physical and functional interaction between Tub4p, Spc98p and Spc97p: first, temperature-sensitive spc97(ts) mutants are suppressed by high gene dosage of SPC98 or TUB4. Second, Spc97p interacts with Spc98p and Tub4p in the two-hybrid system. Finally, immunoprecipitation and fractionation studies revealed complexes containing Tub4p, Spc98p and Spc97p. Further support for a direct interaction of Tub4p, Spc98p and Spc97p comes from the toxicity of strong SPC97 overexpression which is suppressed by co-overexpression of TUB4 or SPC98. Analysis of temperature-sensitive spc97(ts) alleles revealed multiple Spindle defects. While spc97-14 cells are either impaired in SPB separation or mitotic Spindle formation, spc97-20 cells show an additional defect in SPB duplication. We discuss a model in which the Tub4p-Spc98p-Spc97p complex is part of the microtubule attachment site at the SPB.
Mark Winey - One of the best experts on this subject based on the ideXlab platform.
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translational control of mps1 links protein synthesis with the initiation of cell division and Spindle Pole body duplication in yeast
bioRxiv, 2020Co-Authors: Heidi M Blank, Mark Winey, Annabel Alonso, Michael PolymenisAbstract:ABSTRACT Protein synthesis underpins cell growth and controls when cells commit to a new round of cell division at a point in late G1 of the cell cycle called Start. Passage through Start also coincides with the duplication of the microtubule-organizing centers, the yeast Spindle Pole bodies, which will form the two Poles of the mitotic Spindle that segregates the chromosomes in mitosis. The conserved Mps1p kinase governs the duplication of the Spindle Pole body in Saccharomyces cerevisiae. Here, we show that the MPS1 transcript has a short upstream open reading frame that represses the synthesis of Mps1p. Mutating the MPS1 uORF makes the cells smaller, accelerates the appearance of Mps1p in late G1, and promotes completion of Start. The accelerated Start of MPS1 uORF mutants depends on the G1 cyclin Cln3p. Monitoring the Spindle Pole body in the cell cycle using structured illumination microscopy revealed that mutating the MPS1 uORF enabled cells to duplicate their Spindle Pole body earlier, at a smaller cell size. For the first time, these results identify growth inputs in mechanisms that control duplication of the microtubule-organizing center and implicate these processes in general mechanisms that couple growth with division.
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structure function analysis of the c terminal domain of cnm67 a core component of the saccharomyces cerevisiae Spindle Pole body
Journal of Biological Chemistry, 2011Co-Authors: Vadim A Klenchin, Jeremiah J Frye, Michelle Jones, Mark Winey, Ivan RaymentAbstract:Abstract The Spindle Pole body of the budding yeast Saccharomyces cerevisiae has served as a model system for understanding microtubule organizing centers, yet very little is known about the molecular structure of its components. We report here the structure of the C-terminal domain of the core component Cnm67 at 2.3 A resolution. The structure determination was aided by a novel approach to crystallization of proteins containing coiled-coils that utilizes globular domains to stabilize the coiled-coils. This enhances their solubility in Escherichia coli and improves their crystallization. The Cnm67 C-terminal domain (residues Asn-429—Lys-581) exhibits a previously unseen dimeric, interdigitated, all α-helical fold. In vivo studies demonstrate that this domain alone is able to localize to the Spindle Pole body. In addition, the structure reveals a large functionally indispensable positively charged surface patch that is implicated in Spindle Pole body localization. Finally, the C-terminal eight residues are disordered but are critical for protein folding and structural stability.
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the budding yeast Spindle Pole body structure duplication and function
Annual Review of Cell and Developmental Biology, 2004Co-Authors: Sue L Jaspersen, Mark WineyAbstract:▪ Abstract Nucleation of microtubules by eukaryotic microtubule organizing centers (MTOCs) is required for a variety of functions, including chromosome segregation during mitosis and meiosis, cytokinesis, fertilization, cellular morphogenesis, cell motility, and intracellular trafficking. Analysis of MTOCs from different organisms shows that the structure of these organelles is widely varied even though they all share the function of microtubule nucleation. Despite their morphological diversity, many components and regulators of MTOCs, as well as principles in their assembly, seem to be conserved. This review focuses on one of the best-characterized MTOCs, the budding yeast Spindle Pole body (SPB). We review what is known about its structure, protein composition, duplication, regulation, and functions. In addition, we discuss how studies of the yeast SPB have aided investigation of other MTOCs, most notably the centrosome of animal cells.
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multi step control of Spindle Pole body duplication by cyclin dependent kinase
Nature Cell Biology, 2001Co-Authors: Steven B Haase, Mark Winey, Steven I ReedAbstract:Organelles called centrosomes in metazoans or Spindle Pole bodies (SPBs) in yeast direct the assembly of a bipolar Spindle that is essential for faithful segregation of chromosomes during mitosis. Abnormal accumulation of multiple centrosomes leads to genome instability, and has been observed in both tumour cells and cells with targeted mutations in tumour-suppressor genes. The defects that lead to centrosome amplification are not understood. We have recapitulated the multiple-centrosome phenotype in budding yeast by disrupting the activity of specific cyclin-dependent kinase (CDK) complexes. Our observations are reminiscent of mechanisms that govern DNA replication, and show that specific cyclin/CDK activities function both to promote SPB duplication and to prevent SPB reduplication.
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Mechanisms of genetic instability revealed by analysis of yeast Spindle Pole body duplication.
Biology of the Cell, 1999Co-Authors: Heidi J. Chial, Mark WineyAbstract:Aneuploidy and polyploidy are commonly observed in transformed cells. These states arise from failures during mitotic chromosome segregation, some of which can be traced to defects in the function or duplication of the centrosome. The centrosome is the organizing center for the mitotic Spindle, and the equivalent organelle in the budding yeast, Saccharomyces cerevisiae, is the Spindle Pole body. We review how defects in Spindle Pole body duplication or function lead to genetic instability in yeast. There are several well documented instances of genetic instability in yeast that can be traced to the Spindle Pole body, all of which serve as models for genetic instability in transformed cells.
Chikashi Shimoda - One of the best experts on this subject based on the ideXlab platform.
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s pombe sporulation specific coiled coil protein spo15p is localized to the Spindle Pole body and essential for its modification
Journal of Cell Science, 2000Co-Authors: Shigeaki Ikemoto, Michiko Kubo, Taro Nakamura, Chikashi ShimodaAbstract:Spindle Pole bodies in the fission yeast Schizosaccharomyces pombe are required during meiosis, not only for Spindle formation but also for the assembly of forespore membranes. The spo15 mutant is defective in the formation of forespore membranes, which develop into spore envelopes. The spo15 + gene encodes a protein with a predicted molecular mass of 223 kDa, containing potential coiled-coil regions. The spo15 gene disruptant was not lethal, but was defective in spore formation. Northern and western analyses indicated that spo15 + was expressed not only in meiotic cells but also in vegetative cells. When the spo15-GFP fusion gene was expressed by the authentic spo15 promoter during vegetative growth and sporulation, the fusion protein colocalized with Sad1p, which is a component of Spindle Pole bodies. Meiotic divisions proceeded in spo15Δ cells with kinetics similar to those in wild-type cells. In addition, the morphology of the mitotic and meiotic Spindles and the nuclear segregation were normal in spo15Δ. Intriguingly, transformation of Spindle Pole bodies from a punctate to a crescent form prior to forespore membrane formation was not observed in spo15Δ cells. We conclude that Spo15p is associated with Spindle Pole bodies throughout the life cycle and plays an indispensable role in the initiation of spore membrane formation. SUMMARY
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S. pombe sporulation-specific coiled-coil protein Spo15p is localized to the Spindle Pole body and essential for its modification.
Journal of Cell Science, 2000Co-Authors: Shigeaki Ikemoto, Michiko Kubo, Taro Nakamura, Chikashi ShimodaAbstract:Spindle Pole bodies in the fission yeast Schizosaccharomyces pombe are required during meiosis, not only for Spindle formation but also for the assembly of forespore membranes. The spo15 mutant is defective in the formation of forespore membranes, which develop into spore envelopes. The spo15(+)gene encodes a protein with a predicted molecular mass of 223 kDa, containing potential coiled-coil regions. The spo15 gene disruptant was not lethal, but was defective in spore formation. Northern and western analyses indicated that spo15(+) was expressed not only in meiotic cells but also in vegetative cells. When the spo15-GFP fusion gene was expressed by the authentic spo15 promoter during vegetative growth and sporulation, the fusion protein colocalized with Sad1p, which is a component of Spindle Pole bodies. Meiotic divisions proceeded in spo15delta cells with kinetics similar to those in wild-type cells. In addition, the morphology of the mitotic and meiotic Spindles and the nuclear segregation were normal in spo15delta. Intriguingly, transformation of Spindle Pole bodies from a punctate to a crescent form prior to forespore membrane formation was not observed in spo15delta cells. We conclude that Spo15p is associated with Spindle Pole bodies throughout the life cycle and plays an indispensable role in the initiation of spore membrane formation.
Trisha N. Davis - One of the best experts on this subject based on the ideXlab platform.
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novel phosphorylation states of the yeast Spindle Pole body
Biology Open, 2018Co-Authors: Kimberly K Fong, Alex Zelter, Jill M Hoyt, Michael Riffle, Richard S Johnson, Beth Graczyk, Michael J Maccoss, Trisha N. DavisAbstract:ABSTRACT Phosphorylation regulates yeast Spindle Pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of enriched Saccharomyces cerevisiae SPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by a cdc4-1 temperature-sensitive mutation, with particular focus on phosphorylation events on the γ-tubulin small complex (γ-TuSC). The cdc4-1 arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating γ-TuSC. These sites were analyzed in vivo by fluorescence microscopy and were shown to be required for proper regulation of Spindle length. Additionally, in vivo analysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is required for progression through the cell cycle. This phosphoproteomic data set not only broadens the scope of the phosphoproteome of SPBs, it also identifies several γ-TuSC phosphorylation sites that influence microtubule formation.
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novel phosphorylation states of the yeast Spindle Pole body
bioRxiv, 2018Co-Authors: Kimberly K Fong, Alex Zelter, Jill M Hoyt, Michael Riffle, Richard S Johnson, Beth Graczyk, Michael J Maccoss, Trisha N. DavisAbstract:Phosphorylation regulates yeast Spindle Pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of purified Saccharomyces cerevisiae SPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by a cdc4-1 temperature sensitive mutation, with particular interest in phosphorylation events on the γ-tubulin small complex (γ-TuSC). The cdc4-1 arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating γ-TuSC. These sites were analyzed in vivo by fluorescence microscopy and were shown to be required for proper regulation of Spindle length. Additionally, in vivo analysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is required for progression through the cell cycle. This phosphoproteomic data set not only broadens the scope of the phosphoproteome of SPBs, it also identifies several γ-TuSC phosphorylation sites influencing microtubule regulation.
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Purification of Fluorescently Labeled Saccharomyces cerevisiae Spindle Pole Bodies.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Kimberly K Fong, Beth Graczyk, Trisha N. DavisAbstract:Centrosomes are components of the mitotic Spindle responsible for organizing microtubules and establishing a bipolar Spindle for accurate chromosome segregation. In budding yeast, Saccharomyces cerevisiae, the centrosome is called the Spindle Pole body, a highly organized trilaminar structure embedded in the nuclear envelope. Here we describe a detailed protocol for the purification of fluorescently labeled Spindle Pole bodes from S. cerevisiae. Spindle Pole bodies are purified from yeast using a TAP-tag purification followed by velocity sedimentation.This highly reproducible TAP-tag purification method improves upon previous techniques and expands the scope of in vitro characterization of yeast Spindle Pole bodies. The genetic flexibility of this technique allows for the study of Spindle Pole body mutants as well as the study of Spindle Pole bodies during different stages of the cell cycle. The ease and reproducibility of the technique make it possible to study Spindle Pole bodies using a variety of biochemical, biophysical, and microscopic techniques.
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identification of saccharomyces cerevisiae Spindle Pole body remodeling factors
PLOS ONE, 2010Co-Authors: Kristen B Greenland, Huiming Ding, Michael Costanzo, Charles Boone, Trisha N. DavisAbstract:The Saccharomyces cerevisiae centrosome or Spindle Pole body (SPB) is a dynamic structure that is remodeled in a cell cycle dependent manner. The SPB increases in size late in the cell cycle and during most cell cycle arrests and exchanges components during G1/S. We identified proteins involved in the remodeling process using a strain in which SPB remodeling is conditionally induced. This strain was engineered to express a modified SPB component, Spc110, which can be cleaved upon the induction of a protease. Using a synthetic genetic array analysis, we screened for genes required only when Spc110 cleavage is induced. Candidate SPB remodeling factors fell into several functional categories: mitotic regulators, microtubule motors, protein modification enzymes, and nuclear pore proteins. The involvement of candidate genes in SPB assembly was assessed in three ways: by identifying the presence of a synthetic growth defect when combined with an Spc110 assembly defective mutant, quantifying growth of SPBs during metaphase arrest, and comparing distribution of SPB size during asynchronous growth. These secondary screens identified four genes required for SPB remodeling: NUP60, POM152, and NCS2 are required for SPB growth during a mitotic cell cycle arrest, and UBC4 is required to maintain SPB size during the cell cycle. These findings implicate the nuclear pore, urmylation, and ubiquitination in SPB remodeling and represent novel functions for these genes.
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the essential mitotic target of calmodulin is the 110 kilodalton component of the Spindle Pole body in saccharomyces cerevisiae
Molecular and Cellular Biology, 1993Co-Authors: John R Geiser, Holly A Sundberg, Bill H Chang, Eric G D Muller, Trisha N. DavisAbstract:Two independent methods identified the Spindle Pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of Spindle Pole body components localized calmodulin to the Spindle Pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.
John V Kilmartin - One of the best experts on this subject based on the ideXlab platform.
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Spindle Pole body duplication a model for centrosome duplication
Trends in Cell Biology, 2000Co-Authors: Ian R. Adams, John V KilmartinAbstract:The yeast Spindle Pole body (SPB) is the functional equivalent of the centrosome and forms the two Poles of the mitotic Spindle. Before mitosis, both SPBs and centrosomes are present as single copies and must be duplicated to form the bipolar Spindle. SPB components have been identified using a combination of biochemistry and genetics, and their role during SPB duplication has been analysed using temperature-sensitive mutants. In this article, we describe structural aspects of SPB duplication and their possible relationship to centrosome duplication.
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Analysis of the Saccharomyces Spindle Pole by Matrix-assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry
The Journal of cell biology, 1998Co-Authors: Philip A. Wigge, Ole Nørregaard Jensen, Simon Holmes, Sylvie Souès, Matthias Mann, John V KilmartinAbstract:A highly enriched Spindle Pole preparation was prepared from budding yeast and fractionated by SDS gel electrophoresis. Forty-five of the gel bands that appeared enriched in this fraction were analyzed by high-mass accuracy matrix-assisted laser desorption/ ionization (MALDI) peptide mass mapping combined with sequence database searching. This identified twelve of the known Spindle Pole components and an additional eleven gene products that had not previously been localized to the Spindle Pole. Immunoelectron microscopy localized eight of these components to different parts of the Spindle. One of the gene products, Ndc80p, shows homology to human HEC protein (Chen, Y., D.J. Riley, P-L. Chen, and W-H. Lee. 1997. Mol. Cell Biol. 17:6049–6056) and temperature-sensitive mutants show defects in chromosome segregation. This is the first report of the identification of the components of a large cellular organelle by MALDI peptide mapping alone.
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analysis of the saccharomyces Spindle Pole by matrix assisted laser desorption ionization maldi mass spectrometry
Journal of Cell Biology, 1998Co-Authors: Philip A. Wigge, Ole Nørregaard Jensen, Simon Holmes, Sylvie Souès, Matthias Mann, John V KilmartinAbstract:A highly enriched Spindle Pole preparation was prepared from budding yeast and fractionated by SDS gel electrophoresis. Forty-five of the gel bands that appeared enriched in this fraction were analyzed by high-mass accuracy matrix-assisted laser desorption/ ionization (MALDI) peptide mass mapping combined with sequence database searching. This identified twelve of the known Spindle Pole components and an additional eleven gene products that had not previously been localized to the Spindle Pole. Immunoelectron microscopy localized eight of these components to different parts of the Spindle. One of the gene products, Ndc80p, shows homology to human HEC protein (Chen, Y., D.J. Riley, P-L. Chen, and W-H. Lee. 1997. Mol. Cell Biol. 17:6049–6056) and temperature-sensitive mutants show defects in chromosome segregation. This is the first report of the identification of the components of a large cellular organelle by MALDI peptide mapping alone.
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the yeast Spindle Pole body is assembled around a central crystal of spc42p
Cell, 1997Co-Authors: Esther Bullitt, John V Kilmartin, Michael P Rout, Christopher W AkeyAbstract:Abstract The Spindle Pole body (SPB) is the microtubule organizing center (MTOC) in the yeast Saccharomyces that plays a pivotal role in such diverse processes as mitosis, budding, and mating. We have used cryoelectron microscopy and image processing to study the structure of isolated diploid SPBs. We show that SPBs are present in two lateral-size classes, sharing a similar vertical architecture comprised of six major layers. Tomographic reconstructions of heparin-stripped SPBs reveal a central hexagonally packed layer. Overexpression of Spc42p results in the growth of a similar layer, forming a crystal that encircles the SPB. Hence, the SPB is an MTOC that utilizes crystallographic packing of subunits in its construction.
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spc42p a phosphorylated component of the s cerevisiae Spindle Pole body spd with an essential function during spb duplication
Journal of Cell Biology, 1996Co-Authors: A D Donaldson, John V KilmartinAbstract:The 42-kD component of the S. cerevisiae Spindle Pole body (SPB) localizes to the electron-dense central plaque of the SPB. We have cloned the corresponding gene SPC42 (Spindle Pole component) and show that it is essential. Seven temperature-sensitive (ts) mutants in SPC42 were prepared by error-prone PCR. We found that a change to a proline residue in a potential coiled-coil region of Spc42p was responsible for the ts phenotype in at least three alleles, suggesting that formation of the coiled-coil is essential in normal function. The mutant cells showed a phenotype of predominantly single or bilobed SPBs often with an accumulation of unstructured electron-dense material associated with the bridge structure adjacent to the SPB. This phenotype suggests a defect in SPB duplication. This was confirmed by examining synchronized mutant cells that lose viability when SPB duplication is attempted. Spc42p is a phosphoprotein which shows some cell cycle-regulated phosphorylation. Overexpression of Spc42p causes the formation of a disc- or dome-shaped polymer composed of phosphorylated Spc42p, which is attached to the central plaque and associated with the outer nuclear membrane. Taken together, these data suggest that Spc42p forms a polymeric layer at the periphery of the SPB central plaque which has an essential function during SPB duplication and may facilitate attachment of the SPB to the nuclear membrane.
