scholarly journals Localization of the Kar3 kinesin heavy chain-related protein requires the Cik1 interacting protein

1994 ◽  
Vol 124 (4) ◽  
pp. 507-519 ◽  
Author(s):  
BD Page ◽  
LL Satterwhite ◽  
MD Rose ◽  
M Snyder
Keyword(s):  
Heavy Chain ◽  
Fusion Proteins ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Related Protein ◽  
Phenotypic Analysis ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Pole Body ◽  
Kinesin Heavy Chain

The Kar3 protein (Kar3p), a protein related to kinesin heavy chain, and the Cik1 protein (Cik1p) appear to participate in the same cellular processes in S. cerevisiae. Phenotypic analysis of mutants indicates that both CIK1 and KAR3 participate in spindle formation and karyogamy. In addition, the expression of both genes is induced by pheromone treatment. In vegetatively growing cells, both Cik1::beta-gal and Kar3::beta-gal fusions localize to the spindle pole body (SPB), and after pheromone treatment both fusion proteins localize to the spindle pole body and cytoplasmic microtubules. The dependence of Cik1p and Kar3p localization upon one another was investigated by indirect immunofluorescence of fusion proteins in pheromone-treated cells. The Cik1p::beta-gal fusion does not localize to the SPB or microtubules in a kar3 delta strain, and the Kar3p::beta-gal fusion protein does not localize to microtubule-associated structures in a cik1 delta strain. Thus, these proteins appear to be interdependent for localization to the SPB and microtubules. Analysis by both the two-hybrid system and co-immunoprecipitation experiments indicates that Cik1p and kar3p interact, suggesting that they are part of the same protein complex. These data indicate that interaction between a putative kinesin heavy chain-related protein and another protein can determine the localization of motor activity and thereby affect the functional specificity of the motor complex.

scholarly journals Key phosphorylation events in Spc29 and Spc42 guide multiple steps of yeast centrosome duplication

2018 ◽  
Vol 29 (19) ◽  
pp. 2280-2291 ◽  
Author(s):  
Michele Haltiner Jones ◽  
Eileen T. O’Toole ◽  
Amy S. Fabritius ◽  
Eric G. Muller ◽  
Janet B. Meehl ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Centrosome Duplication ◽  
Phosphorylation Sites ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Pole Body ◽  
Core Components ◽  
Spindle Elongation

Phosphorylation modulates many cellular processes during cell cycle progression. The yeast centrosome (called the spindle pole body, SPB) is regulated by the protein kinases Mps1 and Cdc28/Cdk1 as it nucleates microtubules to separate chromosomes during mitosis. Previously we completed an SPB phosphoproteome, identifying 297 sites on 17 of the 18 SPB components. Here we describe mutagenic analysis of phosphorylation events on Spc29 and Spc42, two SPB core components that were shown in the phosphoproteome to be heavily phosphorylated. Mutagenesis at multiple sites in Spc29 and Spc42 suggests that much of the phosphorylation on these two proteins is not essential but enhances several steps of mitosis. Of the 65 sites examined on both proteins, phosphorylation of the Mps1 sites Spc29-T18 and Spc29-T240 was shown to be critical for function. Interestingly, these two sites primarily influence distinct successive steps; Spc29-T240 is important for the interaction of Spc29 with Spc42, likely during satellite formation, and Spc29-T18 facilitates insertion of the new SPB into the nuclear envelope and promotes anaphase spindle elongation. Phosphorylation sites within Cdk1 motifs affect function to varying degrees, but mutations only have significant effects in the presence of an MPS1 mutation, supporting a theme of coregulation by these two kinases.

Identification of an Spc110p-related protein in vertebrates

1997 ◽  
Vol 110 (20) ◽  
pp. 2533-2545 ◽  
Author(s):  
A.M. Tassin ◽  
C. Celati ◽  
M. Paintrand ◽  
M. Bornens
Keyword(s):  
Mammalian Cells ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Partial Purification ◽  
Related Protein ◽  
Microtubule Nucleation ◽  
Pole Body ◽  
Egg Extract ◽  
Ring Complex ◽  
Gamma Tubulin

Although varying in size and complexity, centrosomes have conserved functions throughout the evolutionary range of eukaryotes, and thus may display conserved components. In this work, we took advantage of the recent advances in the isolation of the budding yeast spindle pole body, the development of specific immunological probes and the molecular characterisation of genes involved in spindle pole body duplication or assembly. Screening a monoclonal antibody library against Saccharomyces cerevisiae spindle pole body components, we found that two monoclonal antibodies, directed against two different parts of the yeast Spc110p, decorate the centrosome from mammalian cells in an asymmetrical manner. Western blot experiments identified a 100 kDa protein specifically enriched in centrosome preparations from human cells. This protein is phosphorylated during mitosis and is tightly associated with the centrosome: only denaturing conditions such as 8 M urea were able to solubilise it. Purified immunoglobulins directed against Spc110p inhibit microtubule nucleation on isolated human centrosomes, using brain phosphocellulose-tubulin or Xenopus egg extract tubulin. This result suggested that the centrosomal 100 kDa protein could be involved in a microtubule nucleation complex. To test this hypothesis, we turned to Xenopus species, in which mAb anti-Spc110p decorated centrosomes from somatic cells and identified a 116 kDa protein in egg extract. We performed a partial purification of the gamma-tubulin-ring complex from egg extract. Sucrose gradient sedimentation, immunoprecipitation and native gels demonstrated that the Xenopus 116 kDa protein and gamma-tubulin were found in the same complex. Altogether, these results suggest the existence of an yeast Spc110-related protein in vertebrate centrosomes which is involved in microtubule nucleation.

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 Generation and characterisation of temperature sensitive mutants of genes encoding the fission yeast spindle pole body

2017 ◽  
Author(s):  
Ngang Heok Tang ◽  
Chii Shyang Fong ◽  
Hirohisa Masuda ◽  
Isabelle Jourdain ◽  
Masashi Yukawa ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Genetic Manipulation ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Cellular Processes ◽  
Temperature Sensitive Mutants ◽  
Pole Body ◽  
Genes Encoding ◽  
Mitosis And Meiosis

The spindle pole body (SPB) in fungi is the equivalent of the animal centrosome. A number of previous studies have identified many, if not all, components of the SPB. The SPB is the structural platform for microtubule nucleation and plays important roles, both in mitosis and meiosis. The SPB is absolutely essential for cell survival and its abnormalities give rise to aberrant cell division and morphogenesis. Therefore, it is crucial to understand how the SPB organises itself and how the functions of individual SPB components are regulated. We report here a procedure to generate temperature sensitive mutants in the fission yeast, Schizosaccharomyces pombe. The approach has proved useful to characterise functions of individual SPB components. This original genetic manipulation is however not restricted to analysis of SPB functions, and can be suited to investigate other cellular processes in S. pombe.

scholarly journals Assembly of Yeast Spindle Pole Body and its Components Revealed by Electron Cryo-Tomography

2018 ◽  
Author(s):  
Sam Li ◽  
Jose-Jesus Fernandez
Keyword(s):  
Cell Cycle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
3D Images ◽  
Averaging Methods ◽  
Cellular Processes ◽  
Pole Body ◽  
Organizing Center ◽  
Subtomogram Averaging

AbstractSpindle pole body (SPB) is the microtubule organizing center (MTOC) in yeast. It plays essential roles during many cellular processes, ranging from mitosis to karyogamy. Here, we used electron cryo-tomography (cryo-ET) and image processing to study SPB and its component purified from the budding yeast. The 3D images and models of SPB at various cell cycle stages were reconstructed by cryo-ET and were analyzed. The results reveal SPB as a cylindrical shaped structure composed of multiple layers. The central layers are arranged with a degree of crystalline order. By using subtomogram averaging methods, we studied the purified “sheet” from over-expressed Spc42p. Our analysis of the SPBs and its components provides new insights into the assembly of this organelle and its cellular function as an MTOC.

scholarly journals Higher-order oligomerization of Spc110p drives γ-tubulin ring complex assembly

2016 ◽  
Vol 27 (14) ◽  
pp. 2245-2258 ◽  
Author(s):  
Andrew S. Lyon ◽  
Geneviève Morin ◽  
Michelle Moritz ◽  
King Clyde B. Yabut ◽  
Tamira Vojnar ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Higher Order ◽  
Cellular Processes ◽  
Pole Body ◽  
Small Complex ◽  
Ring Complex ◽  
The Stability

The microtubule (MT) cytoskeleton plays important roles in many cellular processes. In vivo, MT nucleation is controlled by the γ-tubulin ring complex (γTuRC), a 2.1-MDa complex composed of γ-tubulin small complex (γTuSC) subunits. The mechanisms underlying the assembly of γTuRC are largely unknown. In yeast, the conserved protein Spc110p both stimulates the assembly of the γTuRC and anchors the γTuRC to the spindle pole body. Using a quantitative in vitro FRET assay, we show that γTuRC assembly is critically dependent on the oligomerization state of Spc110p, with higher-order oligomers dramatically enhancing the stability of assembled γTuRCs. Our in vitro findings were confirmed with a novel in vivo γTuSC recruitment assay. We conclude that precise spatial control over MT nucleation is achieved by coupling localization and higher-order oligomerization of the receptor for γTuRC.

scholarly journals Differential Regulation of the Kar3p Kinesin-related Protein by Two Associated Proteins, Cik1p and Vik1p

1999 ◽  
Vol 144 (6) ◽  
pp. 1219-1233 ◽  
Author(s):  
Brendan D. Manning ◽  
Jennifer G. Barrett ◽  
Julie A. Wallace ◽  
Howard Granok ◽  
Michael Snyder
Keyword(s):  
Spindle Pole Body ◽  
Structural Similarity ◽  
Spindle Pole ◽  
Body Region ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Related Protein ◽  
Phenotypic Analysis ◽  
Related Proteins

The mechanisms by which kinesin-related proteins interact with other proteins to carry out specific cellular processes is poorly understood. The kinesin-related protein, Kar3p, has been implicated in many microtubule functions in yeast. Some of these functions require interaction with the Cik1 protein (Page, B.D., L.L. Satterwhite, M.D. Rose, and M. Snyder. 1994. J. Cell Biol. 124:507–519). We have identified a Saccharomyces cerevisiae gene, named VIK1, encoding a protein with sequence and structural similarity to Cik1p. The Vik1 protein is detected in vegetatively growing cells but not in mating pheromone-treated cells. Vik1p physically associates with Kar3p in a complex separate from that of the Kar3p-Cik1p complex. Vik1p localizes to the spindle-pole body region in a Kar3p-dependent manner. Reciprocally, concentration of Kar3p at the spindle poles during vegetative growth requires the presence of Vik1p, but not Cik1p. Phenotypic analysis suggests that Cik1p and Vik1p are involved in different Kar3p functions. Disruption of VIK1 causes increased resistance to the microtubule depolymerizing drug benomyl and partially suppresses growth defects of cik1Δ mutants. The vik1Δ and kar3Δ mutations, but not cik1Δ, partially suppresses the temperature-sensitive growth defect of strains lacking the function of two other yeast kinesin-related proteins, Cin8p and Kip1p. Our results indicate that Kar3p forms functionally distinct complexes with Cik1p and Vik1p to participate in different microtubule-mediated events within the same cell.

scholarly journals Generation and characterisation of temperature sensitive mutants of genes encoding the fission yeast spindle pole body

2017 ◽  
Author(s):  
Ngang Heok Tang ◽  
Chii Shyang Fong ◽  
Hirohisa Masuda ◽  
Isabelle Jourdain ◽  
Masashi Yukawa ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Genetic Manipulation ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Cellular Processes ◽  
Temperature Sensitive Mutants ◽  
Pole Body ◽  
Genes Encoding ◽  
Mitosis And Meiosis

The spindle pole body (SPB) in fungi is the equivalent of the animal centrosome. A number of previous studies have identified many, if not all, components of the SPB. The SPB is the structural platform for microtubule nucleation and plays important roles, both in mitosis and meiosis. The SPB is absolutely essential for cell survival and its abnormalities give rise to aberrant cell division and morphogenesis. Therefore, it is crucial to understand how the SPB organises itself and how the functions of individual SPB components are regulated. We report here a procedure to generate temperature sensitive mutants in the fission yeast, Schizosaccharomyces pombe. The approach has proved useful to characterise functions of individual SPB components. This original genetic manipulation is however not restricted to analysis of SPB functions, and can be suited to investigate other cellular processes in S. pombe.

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