Stu2p: A Microtubule-Binding Protein that Is an Essential Component of the Yeast Spindle Pole Body

Previously we isolated tub2-423, a cold-sensitive allele of the Saccharomyces cerevisiae gene encoding β-tubulin that confers a defect in mitotic spindle function. In an attempt to identify additional proteins that are important for spindle function, we screened for suppressors of the cold sensitivity of tub2-423 and obtained two alleles of a novel gene, STU2. STU2 is an essential gene and encodes a protein whose sequence is similar to proteins identified in a variety of organisms. Stu2p localizes primarily to the spindle pole body (SPB) and to a lesser extent along spindle microtubules. Localization to the SPB is not dependent on the presence of microtubules, indicating that Stu2p is an integral component of the SPB. Stu2p also binds microtubules in vitro. We have localized the microtubule-binding domain of Stu2p to a highly basic 100-amino acid region. This region contains two imperfect repeats; both repeats appear to contribute to microtubule binding to similar extents. These results suggest that Stu2p may play a role in the attachment, organization, and/or dynamics of microtubule ends at the SPB.

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The yeast protein kinase Mps1p is required for assembly of the integral spindle pole body component Spc42p

The Journal of Cell Biology ◽

10.1083/jcb.200111025 ◽

2002 ◽

Vol 156 (3) ◽

pp. 453-465 ◽

Cited By ~ 52

Author(s):

Andrea R. Castillo ◽

Janet B. Meehl ◽

Garry Morgan ◽

Amy Schutz-Geschwender ◽

Mark Winey

Keyword(s):

Protein Kinase ◽

Spindle Pole Body ◽

Spindle Checkpoint ◽

Spindle Pole ◽

Physical Interaction ◽

Gene Encoding ◽

Pole Body ◽

Conditional Allele

Saccharomyces cerevisiae MPS1 encodes an essential protein kinase that has roles in spindle pole body (SPB) duplication and the spindle checkpoint. Previously characterized MPS1 mutants fail in both functions, leading to aberrant DNA segregation with lethal consequences. Here, we report the identification of a unique conditional allele, mps1–8, that is defective in SPB duplication but not the spindle checkpoint. The mutations in mps1-8 are in the noncatalytic region of MPS1, and analysis of the mutant protein indicates that Mps1-8p has wild-type kinase activity in vitro. A screen for dosage suppressors of the mps1-8 conditional growth phenotype identified the gene encoding the integral SPB component SPC42. Additional analysis revealed that mps1-8 exhibits synthetic growth defects when combined with certain mutant alleles of SPC42. An epitope-tagged version of Mps1p (Mps1p-myc) localizes to SPBs and kinetochores by immunofluorescence microscopy and immuno-EM analysis. This is consistent with the physical interaction we detect between Mps1p and Spc42p by coimmunoprecipitation. Spc42p is a substrate for Mps1p phosphorylation in vitro, and Spc42p phosphorylation is dependent on Mps1p in vivo. Finally, Spc42p assembly is abnormal in a mps1-1 mutant strain. We conclude that Mps1p regulates assembly of the integral SPB component Spc42p during SPB duplication.

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The Cdc31p-binding protein Kar1p is a component of the half bridge of the yeast spindle pole body.

The Journal of Cell Biology ◽

10.1083/jcb.128.5.863 ◽

1995 ◽

Vol 128 (5) ◽

pp. 863-877 ◽

Cited By ~ 93

Author(s):

A Spang ◽

I Courtney ◽

K Grein ◽

M Matzner ◽

E Schiebel

Keyword(s):

Cell Cycle ◽

Essential Gene ◽

Binding Protein ◽

Spindle Pole Body ◽

Spindle Pole ◽

Yeast Cells ◽

Vitro Assay ◽

Pole Body ◽

Cell Biol

KAR1 has been identified as an essential gene which is involved in karyogamy of mating yeast cells and in spindle pole body duplication of mitotic cells (Rose, M. D., and G. R. Fink. 1987. Cell. 48:1047-1060). We investigated the cell cycle-dependent localization of the Kar1 protein (Kar1p) and its interaction with other SPB components. Kar1p is associated with the spindle pole body during the entire cell cycle of yeast. Immunoelectron microscopic studies with anti-Kar1p antibodies or with the monoclonal antibody 12CA5 using an epitope-tagged, functional Kar1p revealed that Kar1p is associated with the half bridge or the bridge of the spindle pole body. Cdc31p, a Ca(2+)-binding protein, was previously identified as the first component of the half bridge of the spindle pole body (Spang, A., I. Courtney, U. Fackler, M. Matzner, and E. Schiebel. 1993. J. Cell Biol. 123:405-416). Using an in vitro assay we demonstrate that Cdc31p specifically interacts with a short sequence within the carboxyl terminal half of Kar1p. The potential Cdc31p-binding sequence of Kar1p contains three acidic amino acids which are not found in calmodulin-binding peptides, explaining the different substrate specificities of Cdc31p and calmodulin. Cdc31p was also able to bind to the carboxy terminus of Nuflp/Spc110p, another component of the SPB (Kilmartin, J. V., S. L. Dyos, D. Kershaw, and J. T. Finch. 1993. J. Cell Biol. 123:1175-1184). The association of Kar1p with the spindle pole body was independent of Cdc31p. Cdc31p, on the other hand, was not associated with SPBs of kar1 cells.

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Identification of Ribonucleotide Reductase Protein R1 as an Activator of Microtubule Nucleation in Xenopus Egg Mitotic Extracts

Molecular Biology of the Cell ◽

10.1091/mbc.11.12.4173 ◽

2000 ◽

Vol 11 (12) ◽

pp. 4173-4187 ◽

Cited By ~ 8

Author(s):

Saeko Takada ◽

Takehiko Shibata ◽

Yasushi Hiraoka ◽

Hirohisa Masuda

Keyword(s):

Ribonucleotide Reductase ◽

Large Subunit ◽

Spindle Pole Body ◽

Spindle Pole ◽

Microtubule Nucleation ◽

Pole Body ◽

Recombinant Mouse ◽

Xenopus Egg ◽

Essential Subunit

Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared fromXenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopussperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to γ- and α/β-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.

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Bim1p/Yeb1p Mediates the Kar9p-dependent Cortical Attachment of Cytoplasmic Microtubules

Molecular Biology of the Cell ◽

10.1091/mbc.11.9.2949 ◽

2000 ◽

Vol 11 (9) ◽

pp. 2949-2959 ◽

Cited By ~ 102

Author(s):

Rita K. Miller ◽

Soo-Chen Cheng ◽

Mark D. Rose

Keyword(s):

Fluorescent Protein ◽

Binding Protein ◽

Spindle Pole Body ◽

Attachment Site ◽

Spindle Pole ◽

Cell Cortex ◽

Microtubule Binding ◽

Pole Body ◽

Cytoplasmic Microtubules ◽

Two Hybrid

In Saccharomyces cerevisiae, positioning of the mitotic spindle depends on the interaction of cytoplasmic microtubules with the cell cortex. In this process, cortical Kar9p in the bud acts as a link between the actin and microtubule cytoskeletons. To identify Kar9p-interacting proteins, a two-hybrid screen was conducted with the use of full-length Kar9p as bait, and three genes were identified: BIM1, STU2, andKAR9 itself. STU2 encodes a component of the spindle pole body. Bim1p is the yeast homologue of the human microtubule-binding protein EB1, which is a binding partner to the adenomatous polyposis coli protein involved in colon cancer. Eighty-nine amino acids within the third quarter of Bim1p was sufficient to confer interaction with Kar9p. The two-hybrid interactions were confirmed with the use of coimmunoprecipitation experiments. Genetic analysis placed Bim1p in the Kar9p pathway for nuclear migration. Bim1p was not required for Kar9p's cortical or spindle pole body localization. However, deletion ofBIM1 eliminated Kar9p localization along cytoplasmic microtubules. Furthermore, in the bim1 mutants, the cytoplasmic microtubules no longer intersected the cortical dot of Green Fluorescent Protein–Kar9p. These experiments demonstrate that the interaction of cytoplasmic microtubules with the Kar9p cortical attachment site requires the microtubule-binding protein Bim1p.

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γ-Tubulin plays a key role in inactivating APC/CCdh1 at the G1–S boundary

The Journal of Cell Biology ◽

10.1083/jcb.201203115 ◽

2012 ◽

Vol 198 (5) ◽

pp. 785-791 ◽

Cited By ~ 14

Author(s):

Heather Edgerton-Morgan ◽

Berl R. Oakley

Keyword(s):

Cell Cycle ◽

Spindle Pole Body ◽

Spindle Pole ◽

Cyclin B ◽

Anaphase Promoting Complex ◽

Gene Encoding ◽

Localization Pattern ◽

Pole Body ◽

Dependent Inactivation

A γ-tubulin mutation in Aspergillus nidulans, mipA-D159, causes failure of inactivation of the anaphase-promoting complex/cyclosome (APC/C) in interphase, resulting in failure of cyclin B (CB) accumulation and removal of nuclei from the cell cycle. We have investigated the role of CdhA, the A. nidulans homologue of the APC/C activator protein Cdh1, in γ-tubulin–dependent inactivation of the APC/C. CdhA was not essential, but it targeted CB for destruction in G1, and APC/CCdhA had to be inactivated for the G1–S transition. mipA-D159 altered the localization pattern of CdhA, and deletion of the gene encoding CdhA allowed CB to accumulate in all nuclei in strains carrying mipA-D159. These data indicate that mipA-D159 causes a failure of inactivation of APC/CCdhA at G1–S, perhaps by altering its localization to the spindle pole body, and, thus, that γ-tubulin plays an important role in inactivating APC/CCdhA at this point in the cell cycle.

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Role of Septins in the Orientation of Forespore Membrane Extension during Sporulation in Fission Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.01529-09 ◽

2010 ◽

Vol 30 (8) ◽

pp. 2057-2074 ◽

Cited By ~ 24

Author(s):

Masayuki Onishi ◽

Takako Koga ◽

Aiko Hirata ◽

Taro Nakamura ◽

Haruhiko Asakawa ◽

...

Keyword(s):

Spindle Pole Body ◽

Leading Edge ◽

Spindle Pole ◽

Pole Body ◽

Membrane Extension ◽

Fusion Site ◽

Phosphatidylinositol 4 ◽

Ring Shaped Structure

ABSTRACT During yeast sporulation, a forespore membrane (FSM) initiates at each spindle-pole body and extends to form the spore envelope. We used Schizosaccharomyces pombe to investigate the role of septins during this process. During the prior conjugation of haploid cells, the four vegetatively expressed septins (Spn1, Spn2, Spn3, and Spn4) coassemble at the fusion site and are necessary for its normal morphogenesis. Sporulation involves a different set of four septins (Spn2, Spn5, Spn6, and the atypical Spn7) that does not include the core subunits of the vegetative septin complex. The four sporulation septins form a complex in vitro and colocalize interdependently to a ring-shaped structure along each FSM, and septin mutations result in disoriented FSM extension. The septins and the leading-edge proteins appear to function in parallel to orient FSM extension. Spn2 and Spn7 bind to phosphatidylinositol 4-phosphate [PtdIns(4)P] in vitro, and PtdIns(4)P is enriched in the FSMs, suggesting that septins bind to the FSMs via this lipid. Cells expressing a mutant Spn2 protein unable to bind PtdIns(4)P still form extended septin structures, but these structures fail to associate with the FSMs, which are frequently disoriented. Thus, septins appear to form a scaffold that helps to guide the oriented extension of the FSM.

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The microtubule polymerase Stu2 promotes oligomerization of the γ-TuSC for cytoplasmic microtubule nucleation

eLife ◽

10.7554/elife.39932 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 24

Author(s):

Judith Gunzelmann ◽

Diana Rüthnick ◽

Tien-chen Lin ◽

Wanlu Zhang ◽

Annett Neuner ◽

...

Keyword(s):

Budding Yeast ◽

Spindle Pole Body ◽

Family Members ◽

Spindle Pole ◽

Microtubule Nucleation ◽

Cytoplasmic Microtubule ◽

Pole Body ◽

Cytoplasmic Microtubules

Stu2/XMAP215/ZYG-9/Dis1/Alp14/Msps/ch-TOG family members in association with with γ-tubulin complexes nucleate microtubules, but we know little about the interplay of these nucleation factors. Here, we show that the budding yeast Stu2 in complex with the γ-tubulin receptor Spc72 nucleates microtubules in vitro without the small γ-tubulin complex (γ-TuSC). Upon γ-TuSC addition, Stu2 facilitates Spc72–γ-TuSC interaction by binding to Spc72 and γ-TuSC. Stu2 together with Spc72–γ-TuSC increases microtubule nucleation in a process that is dependent on the TOG domains of Stu2. Importantly, these activities are also important for microtubule nucleation in vivo. Stu2 stabilizes Spc72–γ-TuSC at the minus end of cytoplasmic microtubules (cMTs) and an in vivo assay indicates that cMT nucleation requires the TOG domains of Stu2. Upon γ-tubulin depletion, we observed efficient cMT nucleation away from the spindle pole body (SPB), which was dependent on Stu2. Thus, γ-TuSC restricts cMT assembly to the SPB whereas Stu2 nucleates cMTs together with γ-TuSC and stabilizes γ-TuSC at the cMT minus end.

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Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p

The Journal of Cell Biology ◽

10.1083/jcb.200208169 ◽

2002 ◽

Vol 159 (6) ◽

pp. 945-956 ◽

Cited By ~ 119

Author(s):

Sue L. Jaspersen ◽

Thomas H. Giddings ◽

Mark Winey

Keyword(s):

Mitotic Spindle ◽

Spindle Pole Body ◽

Integral Membrane Protein ◽

Spindle Pole ◽

Temperature Sensitive ◽

Nonpermissive Temperature ◽

Reading Frame ◽

Pole Body ◽

Monopolar Spindle

Accurate duplication of the Saccharomyces cerevisiae spindle pole body (SPB) is required for formation of a bipolar mitotic spindle. We identified mutants in SPB assembly by screening a temperature-sensitive collection of yeast for defects in SPB incorporation of a fluorescently marked integral SPB component, Spc42p. One SPB assembly mutant contained a mutation in a previously uncharacterized open reading frame that we call MPS3 (for monopolar spindle). mps3-1 mutants arrest in mitosis with monopolar spindles at the nonpermissive temperature, suggesting a defect in SPB duplication. Execution point experiments revealed that MPS3 function is required for the first step of SPB duplication in G1. Like cells containing mutations in two other genes required for this step of SPB duplication (CDC31 and KAR1), mps3-1 mutants arrest with a single unduplicated SPB that lacks an associated half-bridge. MPS3 encodes an essential integral membrane protein that localizes to the SPB half-bridge. Genetic interactions between MPS3 and CDC31 and binding of Cdc31p to Mps3p in vitro, as well as the fact that Cdc31p localization to the SPB is partially dependent on Mps3p function, suggest that one function for Mps3p during SPB duplication is to recruit Cdc31p, the yeast centrin homologue, to the half-bridge.

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NDC10: a gene involved in chromosome segregation in Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.121.3.503 ◽

1993 ◽

Vol 121 (3) ◽

pp. 503-512 ◽

Cited By ~ 162

Author(s):

P Y Goh ◽

J V Kilmartin

Keyword(s):

Chromosome Segregation ◽

Staining Pattern ◽

Essential Gene ◽

Spindle Pole Body ◽

Spindle Pole ◽

Polyploid Cell ◽

Temperature Sensitive ◽

Sensitive Mutant ◽

Pole Body

A mutant, ndc10-1, was isolated by anti-tubulin staining of temperature-sensitive mutant banks of budding yeast. ndc10-1 has a defect chromosome segregation since chromosomes remains at one pole of the anaphase spindle. This produces one polyploid cell and one aploid cell, each containing a spindle pole body (SPD. NDC10 was cloned and sequenced and is identical to CBF2 (Jiang, W., J. Lechnermn and J. Carbon. 1993. J. Cell Biol. 121:513) which is the 110-kD component of a centromere DNA binding complex (Lechner, J., and J. Carbon. 1991. Cell. 61:717-725). NDC10 is an essential gene. Antibodies to Ndc10p labeled the SPB region in nearly all the cells examined including nonmitotic cells. In some cells with short spindles which may be in metaphase, staining was also observed along the spindle. The staining pattern and the phenotype of ndc10-1 are consistent with Cbf2p/Ndc10p being a kinetochore protein, and provide in vivo evidence for its role in the attachment of chromosomes to the spindle.

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