scholarly journals New Alleles of the YeastMPS1Gene Reveal Multiple Requirements in Spindle Pole Body Duplication

1998 ◽  
Vol 9 (4) ◽  
pp. 759-774 ◽  
Author(s):  
Amy R. Schutz ◽  
Mark Winey
Keyword(s):  
Protein Kinase ◽  
Spindle Assembly Checkpoint ◽  
Single Point ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Pole Body

In Saccharomyces cerevisiae, the Mps1p protein kinase is critical for both spindle pole body (SPB) duplication and the mitotic spindle assembly checkpoint. The mps1–1mutation causes failure early in SPB duplication, and because the spindle assembly checkpoint is also compromised, mps1–1cells proceed with a monopolar mitosis and rapidly lose viability. Here we report the genetic and molecular characterization ofmps1–1 and five new temperature-sensitive alleles ofMPS1. Each of the six alleles contains a single point mutation in the region of the gene encoding the protein kinase domain. The mutations affect several residues conserved among protein kinases, most notably the invariant glutamate in subdomain III. In vivo and in vitro kinase activity of the six epitope-tagged mutant proteins varies widely. Only two display appreciable in vitro activity, and interestingly, this activity is not thermolabile under the assay conditions used. While five of the six alleles cause SPB duplication to fail early, yielding cells with a single SPB, mps1–737cells proceed into SPB duplication and assemble a second SPB that is structurally defective. This phenotype, together with the observation of intragenic complementation between this unique allele and two others, suggests that Mps1p is required for multiple events in SPB duplication.

scholarly journals A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

2017 ◽  
Vol 28 (25) ◽  
pp. 3647-3659 ◽  
Author(s):  
Masashi Yukawa ◽  
Tomoki Kawakami ◽  
Masaki Okazaki ◽  
Kazunori Kume ◽  
Ngang Heok Tang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Bipolar Spindle ◽  
Pole Body ◽  
Cross Linker ◽  
Spindle Elongation

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.

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

2002 ◽  
Vol 156 (3) ◽  
pp. 453-465 ◽  
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.

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

2002 ◽  
Vol 159 (6) ◽  
pp. 945-956 ◽  
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.

scholarly journals The Yeast Spindle Pole Body Component Spc72p Interacts with Stu2p and Is Required for Proper Microtubule Assembly

1998 ◽  
Vol 141 (5) ◽  
pp. 1169-1179 ◽  
Author(s):  
Xiaoyue Peter Chen ◽  
Hongwei Yin ◽  
Tim C. Huffaker
Keyword(s):  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Temperature Sensitive ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Spindle Elongation

We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB). Here we report the identification of Spc72p, a protein that interacts with Stu2p. Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts. Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex. Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p. Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo. A temperature-sensitive spc72 mutation causes defects in SPB morphology. In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB. spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur. The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs.

scholarly journals The Yeast Centrin, Cdc31p, and the Interacting Protein Kinase, Kic1p, Are Required for Cell Integrity

1998 ◽  
Vol 143 (3) ◽  
pp. 751-765 ◽  
Author(s):  
Donald S. Sullivan ◽  
Sue Biggins ◽  
Mark D. Rose
Keyword(s):  
Protein Kinase ◽  
Calcium Binding ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Permissive Temperature ◽  
Cell Integrity ◽  
Interacting Protein ◽  
Pole Body

Cdc31p is the yeast homologue of centrin, a highly conserved calcium-binding protein of the calmodulin superfamily. Previously centrins have been implicated only in microtubule-based processes. To elucidate the functions of yeast centrin, we carried out a two-hybrid screen for Cdc31p-interacting proteins and identified a novel essential protein kinase of 1,080 residues, Kic1p (kinase that interacts with Cdc31p). Kic1p is closely related to S. cerevisiae Ste20p and the p-21– activated kinases (PAKs) found in a wide variety of eukaryotic organisms. Cdc31p physically interacts with Kic1p by two criteria; Cdc31p coprecipitated with GST–Kic1p and it bound to GST–Kic1p in gel overlay assays. Furthermore, GST–Kic1p exhibited in vitro kinase activity that was CDC31-dependent. Although kic1 mutants were not defective for spindle pole body duplication, they exhibited a variety of mutant phenotypes demonstrating that Kic1p is required for cell integrity. We also found that cdc31 mutants, previously identified as defective for spindle pole body duplication, exhibited lysis and morphological defects. The cdc31 kic1 double mutants exhibited a drastic reduction in the range of permissive temperature, resulting in a severe lysis defect. We conclude that Kic1p function is dependent upon Cdc31p both in vivo and in vitro. We postulate that Cdc31p is required both for SPB duplication and for cell integrity/morphogenesis, and that the integrity/morphogenesis function is mediated through the Kic1p protein kinase.

scholarly journals The Yeast CDC37 Gene Interacts with MPS1 and Is Required for Proper Execution of Spindle Pole Body Duplication

1997 ◽  
Vol 136 (5) ◽  
pp. 969-982 ◽  
Author(s):  
Amy R. Schutz ◽  
Thomas H. Giddings ◽  
Estelle Steiner ◽  
Mark Winey
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
G1 Arrest ◽  
Multicopy Suppressor ◽  
Protein Levels ◽  
Pole Body ◽  
Synthetic Lethal Interactions ◽  
Sensitive Allele

The MPS1 gene from Saccharomyces cerevisiae encodes an essential protein kinase required for spindle pole body (SPB) duplication and for the mitotic spindle assembly checkpoint. Cells with the mps1-1 mutation fail early in SPB duplication and proceed through monopolar mitosis with lethal consequences. We identified CDC37 as a multicopy suppressor of mps1-1 temperature-sensitive growth. Suppression is allele specific, and synthetic lethal interactions occur between mps1 and cdc37 alleles. We examined the cdc37-1 phenotype for defects related to the SPB cycle. The cdc37-1 temperature-sensitive allele causes unbudded, G1 arrest at Start (Reed, S.I. 1980. Genetics. 95: 561–577). Reciprocal shifts demonstrate that cdc37-1 arrest is interdependent with α-factor arrest but is not a normal Start arrest. Although the cells are responsive to α-factor at the arrest, SPB duplication is uncoupled from other aspects of G1 progression and proceeds past the satellite-bearing SPB stage normally seen at Start. Electron microscopy reveals side-by-side SPBs at cdc37-1 arrest. The outer plaque of one SPB is missing or reduced, while the other is normal. Using the mps2-1 mutation to distinguish between the SPBs, we find that the outer plaque defect is specific to the new SPB. This phenotype may arise in part from reduced Mps1p function: although Mps1p protein levels are unaffected by the cdc37-1 mutation, kinase activity is markedly reduced. These data demonstrate a requirement for CDC37 in SPB duplication and suggest a role for this gene in G1 control. CDC37 may provide a chaperone function that promotes the activity of protein kinases.

scholarly journals Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

2003 ◽  
Vol 162 (7) ◽  
pp. 1211-1221 ◽  
Author(s):  
John V. Kilmartin
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Binding Motif ◽  
Temperature Sensitive ◽  
Pole Body ◽  
Human Proteins ◽  
Z Domain ◽  
Essential Function

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved.

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

1997 ◽  
Vol 139 (5) ◽  
pp. 1271-1280 ◽  
Author(s):  
Peijing Jeremy Wang ◽  
Tim C. Huffaker
Keyword(s):  
Essential Gene ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cold Sensitivity ◽  
Gene Encoding ◽  
Microtubule Binding ◽  
Amino Acid Region ◽  
Pole Body ◽  
Spindle Function

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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