scholarly journals The 110-kD spindle pole body component of Saccharomyces cerevisiae is a phosphoprotein that is modified in a cell cycle-dependent manner.

1996 ◽  
Vol 132 (5) ◽  
pp. 903-914 ◽  
Author(s):  
D B Friedman ◽  
H A Sundberg ◽  
E Y Huang ◽  
T N Davis
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Wild Type ◽  
Spindle Formation ◽  
Pole Body ◽  
A Cell ◽  
In Cells

Spc110p (Nuf1p) is an essential component of the yeast microtubule organizing center, or spindle pole body (SPB). Asynchronous wild-type cultures contain two electrophoretically distinct isoforms of Spc110p as detected by Western blot analysis, suggesting that Spc110p is modified in vivo. Both isoforms incorporate 32Pi in vivo, suggesting that Spc110p is post-translationally modified by phosphorylation. The slower-migrating 120-kD Spc110p isoform after incubation is converted to the faster-migrating 112-kD isoform after incubation with protein phosphatase PP2A, and specific PP2A inhibitors block this conversion. Thus, additional phosphorylation of Spc110p at serine and/or threonine residues gives rise to the slower-migrating 120-kD isoform. The 120-kD isoform predominates in cells arrested in mitosis by the addition of nocodazole. However, the 120-kD isoform is not detectable in cells grown to stationary phase (G0) or in cells arrested in G1 by the addition of alpha-factor. Temperature-sensitive cell division cycle (cdc) mutations demonstrate that the presence of the 120-kD isoform correlates with mitotic spindle formation but not with SPB duplication. In a synchronous wild-type population, the additional serine/threonine phosphorylation that gives rise to the 120-kD isoform appears as cells are forming the mitotic spindle and diminishes as cells enter anaphase. None of several sequences similar to the consensus for phosphorylation by the Cdc28p (cdc2p34) kinase is important for these mitosis-specific phosphorylations or for function. Carboxy-terminal Spc110p truncations lacking the calmodulin binding site can support growth and are also phosphorylated in a cell cycle-specific manner. Further truncation of the Spc110p carboxy terminus results in mutant proteins that are unable to support growth and now migrate as single species. Collectively, these results provide the first evidence of a structural component of the SPB that is phosphorylated during spindle formation and dephosphorylated as cells enter anaphase.

scholarly journals Mutant Membrane Protein of the Budding Yeast Spindle Pole Body Is Targeted to the Endoplasmic Reticulum Degradation Pathway

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 567-578 ◽  
Author(s):  
Susan McBratney ◽  
Mark Winey
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Degradation Pathway ◽  
Spindle Pole ◽  
Wild Type ◽  
Er Quality Control ◽  
Pole Body ◽  
Cytoplasmic Face ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugation

Abstract Mutation of either the yeast MPS2 or the NDC1 gene leads to identical spindle pole body (SPB) duplication defects: The newly formed SPB is improperly inserted into the nuclear envelope (NE), preventing the cell from forming a bipolar mitotic spindle. We have previously shown that both MPS2 and NDC1 encode integral membrane proteins localized at the SPB. Here we show that CUE1, previously known to have a role in coupling ubiquitin conjugation to ER degradation, is an unusual dosage suppressor of mutations in MPS2 and NDC1. Cue1p has been shown to recruit the soluble ubiquitin-conjugating enzyme, Ubc7p, to the cytoplasmic face of the ER membrane where it can ubiquitinate its substrates and target them for degradation by the proteasome. Both mps2-1 and ndc1-1 are also suppressed by disruption of UBC7 or its partner, UBC6. The Mps2-1p mutant protein level is markedly reduced compared to wild-type Mps2p, and deletion of CUE1 restores the level of Mps2-1p to nearly wild-type levels. Our data indicate that Mps2p may be targeted for degradation by the ER quality control pathway.

The Budding Yeast Cdc15 Localizes to the Spindle Pole Body in a Cell-Cycle-Dependent Manner

1999 ◽  
Vol 2 (3) ◽  
pp. 178-184 ◽  
Author(s):  
R. Cenamor ◽  
J. Jiménez ◽  
V.J. Cid ◽  
C. Nombela ◽  
M. Sánchez
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Dependent Manner ◽  
Pole Body ◽  
A Cell ◽  
Cycle Dependent

scholarly journals Novel Phosphorylation States of the Yeast Spindle Pole Body

2018 ◽  
Author(s):  
Kimberly K. Fong ◽  
Alex Zelter ◽  
Beth Graczyk ◽  
Jill M. Hoyt ◽  
Michael Riffle ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Phosphorylation Sites ◽  
Microtubule Nucleation ◽  
Data Set ◽  
Pole Body

ABSTRACTPhosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of purifiedSaccharomyces cerevisiaeSPBs 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 acdc4-1temperature sensitive mutation, with particular interest in phosphorylation events on the γ-tubulin small complex (γ-TuSC). Thecdc4-1arrest 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 analyzedin vivoby fluorescence microscopy and were shown to be required for proper regulation of spindle length. Additionally,in vivoanalysis 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.

scholarly journals Mutational Analysis Reveals a Role for the C Terminus of the Proteasome Subunit Rpt4p in Spindle Pole Body Duplication inSaccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 705-720 ◽  
Author(s):  
Heather B McDonald ◽  
Astrid Hoes Helfant ◽  
Erin M Mahony ◽  
Shaun K Khosla ◽  
Loretta Goetsch
Keyword(s):  
Cell Cycle ◽  
Mutational Analysis ◽  
Genetic Interaction ◽  
Spindle Pole Body ◽  
Coiled Coil ◽  
Spindle Pole ◽  
Terminal Region ◽  
Wild Type ◽  
Pole Body ◽  
C Terminus

AbstractThe ubiquitin/proteasome pathway plays a key role in regulating cell cycle progression. Previously, we reported that a conditional mutation in the Saccharomyces cerevisiae gene RPT4/PCS1, which encodes one of six ATPases in the proteasome 19S cap complex/regulatory particle (RP), causes failure of spindle pole body (SPB) duplication. To improve our understanding of Rpt4p, we created 58 new mutations, 53 of which convert clustered, charged residues to alanine. Virtually all mutations that affect the N-terminal region, which contains a putative nuclear localization signal and coiled-coil motif, result in a wild-type phenotype. Nine mutations that affect the central ATPase domain and the C-terminal region confer recessive lethality. The two conditional mutations identified, rpt4-145 and rpt4-150, affect the C terminus. After shift to high temperature, these mutations generally cause cells to progress slowly through the first cell cycle and to arrest in the second cycle with large buds, a G2 content of DNA, and monopolar spindles, although this phenotype can vary depending on the medium. Additionally, we describe a genetic interaction between RPT4 and the naturally polymorphic gene SSD1, which in wild-type form modifies the rpt4-145 phenotype such that cells arrest in G2 of the first cycle with complete bipolar spindles.

Evidence for cell cycle-specific, spindle pole body-mediated, nuclear positioning in the fission yeast Schizosaccharomyces pombe

1997 ◽  
Vol 110 (16) ◽  
pp. 1851-1866 ◽  
Author(s):  
I. Hagan ◽  
M. Yanagida
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Nuclear Positioning ◽  
Central Location ◽  
Pole Body ◽  
Spindle Pole Bodies ◽  
A Cell ◽  
Multiple Spindle

Specific changes in spatial order occur during cell cycle progression in fission yeast. Growth of the rod-shaped cells is highly regulated and undergoes a cell cycle and size-regulated switch from monopolar to bipolar tip extension. During both phases of growth, the interphase nucleus is maintained in a central location. Following the separation of the genome to the cell tips in mitosis, the two nuclei migrate back towards the cell equator before stopping in two new positions that will become the middle of the two new cells. Here we use simultaneous labeling of microtubules, chromatin and spindle pole bodies in wild-type and cdc mutants, to show that nuclear positioning is achieved by regulation of spindle pole body-mediated nuclear migration. We show that the number and location of nuclear positioning signals is regulated in a cell cycle-specific manner and that spindle pole body-mediated forces are likely to be responsible for maintaining correct nuclear position once the nuclei have reached the appropriate position in the cell. Accentuating the movement of the nuclei back towards the cell equator after mitosis by artificially increasing cell length shows that the spindle pole body leads the nucleus during this migration. When multiple spindle pole bodies are associated with the same or different nuclei they all go to the same point indicating that the different spindle pole bodies are responding to the same positional cue. In a septation-defective mutant cell, which contains four nuclei, the spindle pole bodies on the four different nuclei initially group as two pairs in regions that would become the middle of the new cells, were the cell able to divide. In the subsequent interphase, the nuclei aggregate as a group of four in the centre of the cell. The presence of two or three clusters of spindle pole bodies in larger cells with eight nuclei suggests that the mechanisms specifying the normally central location for multiple nuclei may be unable to operate properly as the cells get larger. Perturbation of microtubules with the microtubule poison thiabendazole prevents the spindle pole body clustering in septation mutants, demonstrating that nuclear positioning requires a functional microtubule cytoskeleton.

γ-Tubulin and the fungal microtubule cytoskeleton

1995 ◽  
Vol 73 (S1) ◽  
pp. 352-358 ◽  
Author(s):  
Berl R. Oakley
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
Pole Body ◽  
Spindle Pole Bodies ◽  
Key Words Tubulin ◽  
Organizing Center ◽  
Chromosomal Condensation

γ-Tubulin is present in phylogenetically diverse eukaryotes. It is a component of microtubule organizing centers such as the spindle pole bodies of fungi. In Aspergillus nidulans and Schizosaccharomyces pombe, it is essential for nuclear division, and, thus, for viability. In A. nidulans, nuclei carrying a γ-tubulin disruption can be maintained in heterokaryons, and the phenotypes caused by the disruption can be determined in uninucleate spores produced by the heterokaryons. Experiments with heterokaryons created in strains with mutations that allow synchronization of the cell cycle reveal that γ-tubulin is not required for the transition from the G1 phase of the cell cycle through S phase to G2, nor for the entry into mitosis as judged by chromosomal condensation. It is, however, required for the formation of the mitotic spindle and for the successful completion of mitosis. Staining with the MPM-2 monoclonal antibody reveals that spindle pole body replication occurs in the absence of functional γ-tubulin. Finally, human γ-tubulin functions in fission yeast, and this indicates that γ-tubulin has similar functions in widely divergent organisms. Key words: tubulin, microtubule, spindle pole body, microtubule organizing center.

scholarly journals Studies concerning the temporal and genetic control of cell polarity in Saccharomyces cerevisiae.

1991 ◽  
Vol 114 (3) ◽  
pp. 515-532 ◽  
Author(s):  
M Snyder ◽  
S Gehrung ◽  
B D Page
Keyword(s):  
Cell Cycle ◽  
Cell Polarity ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Yeast Cells ◽  
Cell Cycle Distribution ◽  
Restrictive Temperature ◽  
Microtubule Bundle ◽  
Pole Body ◽  
Capture Site

The establishment of cell polarity was examined in the budding yeast, S. cerevisiae. The distribution of a polarized protein, the SPA2 protein, was followed throughout the yeast cell cycle using synchronized cells and cdc mutants. The SPA2 protein localizes to a patch at the presumptive bud site of G1 cells. Later it concentrates at the bud tip in budded cells. At cytokinesis, the SPA2 protein is at the neck between the mother and daughter cells. Analysis of unbudded haploid cells has suggested a series of events that occurs during G1. The SPA2 patch is established very early in G1, while the spindle pole body residues on the distal side of the nucleus. Later, microtubules emanating from the spindle pole body intersect the SPA2 crescent, and the nucleus probably rotates towards the SPA2 patch. By middle G1, most cells contain the SPB on the side of the nucleus proximal to the SPA2 patch, and a long extranuclear microtubule bundle intersects this patch. We suggest that a microtubule capture site exists in the SPA2 staining region that stabilizes the long microtubule bundle; this capture site may be responsible for rotation of the nucleus. Cells containing a polarized distribution of the SPA2 protein also possess a polarized distribution of actin spots in the same region, although the actin staining is much more diffuse. Moreover, cdc4 mutants, which form multiple buds at the restrictive temperature, exhibit simultaneous staining of the SPA2 protein and actin spots in a subset of the bud tips. spa2 mutants contain a polarized distribution of actin spots, and act1-1 and act1-2 mutants often contain a polarized distribution of the SPA2 protein suggesting that the SPA2 protein is not required for localization of the actin spots and the actin spots are not required for localization of the SPA2 protein. cdc24 mutants, which fail to form buds at the restrictive temperature, fail to exhibit polarized localization of the SPA2 protein and actin spots, indicating that the CDC24 protein is directly or indirectly responsible for controlling the polarity of these proteins. Based on the cell cycle distribution of the SPA2 protein, a "cytokinesis tag" model is proposed to explain the mechanism of the non-random positioning of bud sites in haploid yeast cells.

scholarly journals Cdk1 promotes cytokinesis in fission yeast through activation of the septation initiation network

2014 ◽  
Vol 25 (15) ◽  
pp. 2250-2259 ◽  
Author(s):  
Nicole Rachfall ◽  
Alyssa E. Johnson ◽  
Sapna Mehta ◽  
Jun-Song Chen ◽  
Kathleen L. Gould
Keyword(s):  
Cell Cycle ◽  
Spindle Pole Body ◽  
Complete Removal ◽  
Spindle Pole ◽  
Cyclin Dependent Kinase ◽  
Gtpase Activating Protein ◽  
Pole Body ◽  
Kinase Cascade ◽  
Septation Initiation Network ◽  
Cycle Dependent

In Schizosaccharomyces pombe, late mitotic events are coordinated with cytokinesis by the septation initiation network (SIN), an essential spindle pole body (SPB)–associated kinase cascade, which controls the formation, maintenance, and constriction of the cytokinetic ring. It is not fully understood how SIN initiation is temporally regulated, but it depends on the activation of the GTPase Spg1, which is inhibited during interphase by the essential bipartite GTPase-activating protein Byr4-Cdc16. Cells are particularly sensitive to the modulation of Byr4, which undergoes cell cycle–dependent phosphorylation presumed to regulate its function. Polo-like kinase, which promotes SIN activation, is partially responsible for Byr4 phosphorylation. Here we show that Byr4 is also controlled by cyclin-dependent kinase (Cdk1)–mediated phosphorylation. A Cdk1 nonphosphorylatable Byr4 phosphomutant displays severe cell division defects, including the formation of elongated, multinucleate cells, failure to maintain the cytokinetic ring, and compromised SPB association of the SIN kinase Cdc7. Our analyses show that Cdk1-mediated phosphoregulation of Byr4 facilitates complete removal of Byr4 from metaphase SPBs in concert with Plo1, revealing an unexpected role for Cdk1 in promoting cytokinesis through activation of the SIN pathway.

The influence of the microtubule inhibitor, methyl benzimidazol-2-yl-carbamate (MBC) on nuclear division and the cell cycle in Saccharomyces cerevisiae

1980 ◽  
Vol 46 (1) ◽  
pp. 341-352
Author(s):  
R.A. Quinlan ◽  
C.I. Pogson ◽  
K. Gull
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Ultrastructural Examination ◽  
Microtubule Inhibitor ◽  
Microtubule Polymerization ◽  
Pole Body ◽  
Outer Component ◽  
Spindle Microtubules

Methyl benzimidazol-2-yl-carbamate (MBC), at a concentration of 100 microM, has a pronounced effect on the growth of Saccharomyces cerevisiae, resulting in the accumulation of cells as large doublets. We have determined a specific execution point for the effect of MBC on the yeast cell cycle, and have shown that this execution point is between the cycle events of spindle pole body duplication and spindle pole body separation. An ultrastructural examination of the MBC-treated cells revealed the absence of cytoplasmic and spindle microtubules. MBC treatment also produced an altered spindle pole body morphology, causing the disappearance of the outer component. Nuclear size was also markedly increased in the MBC-induced doublet cells, although the septa were completely absent from these doublet cells. It is proposed that MBC inhibits microtubule polymerization, rather than causing the depolymerization of stable microtubules.

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