Sme4 coiled-coil protein mediates synaptonemal complex assembly, recombinosome relocalization, and spindle pole body morphogenesis

During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion.

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Nuf2, a spindle pole body-associated protein required for nuclear division in yeast.

The Journal of Cell Biology ◽

10.1083/jcb.125.4.853 ◽

1994 ◽

Vol 125 (4) ◽

pp. 853-866 ◽

Cited By ~ 43

Author(s):

M A Osborne ◽

G Schlenstedt ◽

T Jinks ◽

P A Silver

Keyword(s):

Nuclear Division ◽

Spindle Pole Body ◽

Coiled Coil ◽

Temperature Shift ◽

Spindle Pole ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Culture Cells ◽

Pole Body ◽

Associated Proteins

The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content of potential coiled-coil structure similar to myosin. Nuf2 is associated with the spindle pole body (SPB) as determined by coimmunofluorescence with known SPB proteins. Nuf2 appears to be localized to the intranuclear region and is a candidate for a protein involved in SPB separation. The nuclear association of Nuf2 can be disrupted, in part, by 1 M salt but not by the detergent Triton X-100. All Nuf2 can be removed from nuclei by 8 M urea extraction. In this regard, Nuf2 is similar to other SPB-associated proteins including Nufl/SPC110, also a coiled-coil protein. Temperature-sensitive alleles of NUF2 were generated within the coiled-coil region of Nuf2 and such NUF2 mutant cells rapidly arrest after temperature shift with a single undivided or partially divided nucleus in the bud neck, a shortened mitotic spindle and their DNA fully replicated. In sum, Nuf2 is a protein associated with the SPB that is critical for nuclear division. Anti-Nuf2 antibodies also recognize a mammalian 73-kd protein and display centrosome staining of mammalian tissue culture cells suggesting the presence of a protein with similar function.

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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 ◽

10.1242/jcs.113.3.545 ◽

2000 ◽

Vol 113 (3) ◽

pp. 545-554 ◽

Cited By ~ 8

Author(s):

S. Ikemoto ◽

T. Nakamura ◽

M. Kubo ◽

C. Shimoda

Keyword(s):

Life Cycle ◽

Fusion Gene ◽

Spindle Pole Body ◽

Coiled Coil ◽

Spindle Pole ◽

Wild Type ◽

Membrane Formation ◽

Pole Body ◽

Spindle Pole Bodies ◽

Meiotic Spindles

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.

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Specific coiled-coil interactions contribute to a global model of the structure of the spindle pole body

Journal of Structural Biology ◽

10.1016/j.jsb.2010.01.022 ◽

2010 ◽

Vol 170 (2) ◽

pp. 246-256 ◽

Cited By ~ 6

Author(s):

Nora Zizlsperger ◽

Amy E. Keating

Keyword(s):

Spindle Pole Body ◽

Coiled Coil ◽

Spindle Pole ◽

Global Model ◽

Pole Body

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The molecular architecture of the yeast spindle pole body core determined by Bayesian integrative modeling

Molecular Biology of the Cell ◽

10.1091/mbc.e17-06-0397 ◽

2017 ◽

Vol 28 (23) ◽

pp. 3298-3314 ◽

Cited By ~ 23

Author(s):

Shruthi Viswanath ◽

Massimiliano Bonomi ◽

Seung Joong Kim ◽

Vadim A. Klenchin ◽

Keenan C. Taylor ◽

...

Keyword(s):

Cell Biology ◽

Spindle Pole Body ◽

Coiled Coil ◽

Resonance Energy ◽

Spindle Pole ◽

Physical Structure ◽

Molecular Architecture ◽

X Ray ◽

Pole Body

Microtubule-organizing centers (MTOCs) form, anchor, and stabilize the polarized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and assembles a bipolar spindle during mitosis to capture and segregate sister chromatids. Yet, despite their importance in cell biology, the physical structure of MTOCs is poorly understood. Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in vivo fluorescence resonance energy transfer (FRET), small-angle x-ray scattering (SAXS), x-ray crystallography, electron microscopy, and two-hybrid analysis. The model is validated by several methods that include a genetic analysis of the conserved PACT domain that recruits Spc110, a protein related to pericentrin, to the SPB. The model suggests that calmodulin can act as a protein cross-linker and Spc29 is an extended, flexible protein. The model led to the identification of a single, essential heptad in the coiled-coil of Spc110 and a minimal PACT domain. It also led to a proposed pathway for the integration of Spc110 into the SPB.

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Cnm67p Is a Spacer Protein of theSaccharomyces cerevisiaeSpindle Pole Body Outer Plaque

Molecular Biology of the Cell ◽

10.1091/mbc.12.8.2519 ◽

2001 ◽

Vol 12 (8) ◽

pp. 2519-2533 ◽

Cited By ~ 35

Author(s):

Florian Schaerer ◽

Garry Morgan ◽

Mark Winey ◽

Peter Philippsen

Keyword(s):

Spindle Pole Body ◽

Coiled Coil ◽

Nuclear Migration ◽

Spindle Pole ◽

Thin Sections ◽

Pole Body ◽

C Terminus ◽

Functional Homolog ◽

N Terminus ◽

Terminal Domains

In Saccharomyces cerevisiae, the spindle pole body (SPB) is the functional homolog of the mammalian centrosome, responsible for the organization of the tubulin cytoskeleton. Cytoplasmic (astral) microtubules essential for the proper segregation of the nucleus into the daughter cell are attached at the outer plaque on the SPB cytoplasmic face. Previously, it has been shown that Cnm67p is an integral component of this structure; cells deleted forCNM67 are lacking the SPB outer plaque and thus experience severe nuclear migration defects. With the use of partial deletion mutants of CNM67, we show that the N- and C-terminal domains of the protein are important for nuclear migration. The C terminus, not the N terminus, is essential for Cnm67p localization to the SPB. On the other hand, only the N terminus is subject to protein phosphorylation of a yet unknown function. Electron microscopy of SPB serial thin sections reveals that deletion of the N- or C-terminal domains disturbs outer plaque formation, whereas mutations in the central coiled-coil domain of Cnm67p change the distance between the SPB core and the outer plaque. We conclude that Cnm67p is the protein that connects the outer plaque to the central plaque embedded in the nuclear envelope, adjusting the space between them by the length of its coiled-coil.

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Saccharomyces cerevisiae MPS2Encodes a Membrane Protein Localized at the Spindle Pole Body and the Nuclear Envelope

Molecular Biology of the Cell ◽

10.1091/mbc.10.7.2393 ◽

1999 ◽

Vol 10 (7) ◽

pp. 2393-2406 ◽

Cited By ~ 38

Author(s):

Marı́a de la Cruz Muñoz-Centeno ◽

Susan McBratney ◽

Antonio Monterrosa ◽

Breck Byers ◽

Carl Mann ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Protein ◽

Nuclear Envelope ◽

Fluorescent Protein ◽

Spindle Pole Body ◽

Coiled Coil ◽

Spindle Pole ◽

Yeast Saccharomyces Cerevisiae ◽

Pole Body ◽

Monopolar Spindle

The MPS2 (monopolar spindle two) gene is one of several genes required for the proper execution of spindle pole body (SPB) duplication in the budding yeast Saccharomyces cerevisiae ( Winey et al., 1991 ). We report here that the MPS2 gene encodes an essential 44-kDa protein with two putative coiled-coil regions and a hydrophobic sequence. Although MPS2 is required for normal mitotic growth, some null strains can survive; these survivors exhibit slow growth and abnormal ploidy. The MPS2 protein was tagged with nine copies of the myc epitope, and biochemical fractionation experiments show that it is an integral membrane protein. Visualization of a green fluorescent protein (GFP) Mps2p fusion protein in living cells and indirect immunofluorescence microscopy of 9xmyc-Mps2p revealed a perinuclear localization with one or two brighter foci of staining corresponding to the SPB. Additionally, immunoelectron microscopy shows that GFP-Mps2p localizes to the SPB. Our analysis suggests that Mps2p is required as a component of the SPB for insertion of the nascent SPB into the nuclear envelope.

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

Genetics ◽

10.1093/genetics/162.2.705 ◽

2002 ◽

Vol 162 (2) ◽

pp. 705-720 ◽

Cited By ~ 3

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.

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Meiotic Spindle Pole Bodies Acquire the Ability to Assemble the Spore Plasma Membrane by Sequential Recruitment of Sporulation-specific Components in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e08-02-0118 ◽

2008 ◽

Vol 19 (6) ◽

pp. 2476-2487 ◽

Cited By ~ 23

Author(s):

Yukiko Nakase ◽

Michiko Nakamura-Kubo ◽

Yanfang Ye ◽

Aiko Hirata ◽

Chikashi Shimoda ◽

...

Keyword(s):

De Novo ◽

Close Contact ◽

Spindle Pole Body ◽

Coiled Coil ◽

Spindle Pole ◽

Meiotic Spindle ◽

Deletion Mutants ◽

Pole Body ◽

Spindle Pole Bodies ◽

Cytoplasmic Side

The spindle pole body (SPB) of Schizosaccharomyces pombe is required for assembly of the forespore membrane (FSM) during meiosis. Before de novo biogenesis of the FSM, the meiotic SPB forms outer plaques, an event referred to as SPB modification. A constitutive SPB component, Spo15, plays an indispensable role in SPB modification and sporulation. Here, we analyzed two sporulation-specific genes, spo13+ and spo2+, which are not required for progression of meiotic nuclear divisions, but are essential for sporulation. Spo13 is a 16-kDa coiled-coil protein, and Spo2 is a 15-kDa nonconserved protein. Both Spo13 and Spo2 specifically associated with the meiotic SPB. The respective deletion mutants are viable, but defective in SPB modification and in the onset of FSM formation. Spo13 and Spo2 localized on the cytoplasmic side of the SPB in close contact with the nascent FSM. Localization of Spo13 to the SPB was dependent on Spo15 and Spo2; that of Spo2 depended only on Spo15, suggesting that their recruitment to the SPB is strictly controlled. Spo2 physically associated with both Spo15 and Spo13, but Spo13 and Spo15 did not interact directly. Taken together, these observations indicate that Spo2 is recruited to the SPB during meiosis and then assists in the localization of Spo13 to the outer surface of the SPB.

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