scholarly journals Yeast ubiquitin-like genes are involved in duplication of the microtubule organizing center.

1996 ◽  
Vol 133 (6) ◽  
pp. 1331-1346 ◽  
Author(s):  
S Biggins ◽  
I Ivanovska ◽  
M D Rose
Keyword(s):  
Excision Repair ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Repair Gene ◽  
Nucleotide Excision ◽  
Organizing Center ◽  
Dependent Protein

KAR1 is required for duplication of the Saccharomyces cerevisiae microtubule organizing center, the spindle pole body (SPB) (Rose, M.D., and G.R. Fink, 1987. Cell. 48:1047-1060). Suppressors of a kar1 allele defective for SPB duplication were isolated in two genes, CDC31 and DSK2 (Vallen, E.A., W.H., M. Winey, and M.D. Rose. 1994. Genetics. 137:407-422). To elucidate the role of DSK2 in SPB duplication, we cloned the gene and found it encodes a novel ubiquitin-like protein containing an NH2 terminus 36% identical to ubiquitin. The only other known yeast ubiquitin-like protein is encoded by the nucleotide excision repair gene RAD23 (Watkins, J.F.,P. Sung, L. Prakash, and S. Prakash. 1993. Mol. Cell. Bio. 13:7757-7765). Unlike ubiquitin, the NH2-terminal domain of Dsk2p is not cleaved from the protein, indicating that Dsk2p is not conjugated to other proteins. Although the DSK2-1 mutation alters a conserved residue in the Dsk2p ubiquitin-like domain, we detect no differences in Dsk2p or Cdc31p stability. Therefore, DSK2 does not act by interfering with ubiquitin-dependent protein degradation of these proteins. Although DSK2 is not essential, a strain deleted for both DSK2 and RAD23 is temperature sensitive for growth due to a block in SPB duplication. In addition, overexpression of DSK2 is toxic, and the DSK2-1 allele causes a block in SPB duplication. Therefore, DSK2 dosage is critical for SPB duplication. We determined that CDC31 gene function is downstream of DSK2 and KAR1. Dsk2p is a nuclear-enriched protein, and we propose that Dsk2p assists in Cdc31 assembly into the new SPB.

Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organizing center in Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 137 (2) ◽  
pp. 407-422 ◽  
Author(s):  
E A Vallen ◽  
W Ho ◽  
M Winey ◽  
M D Rose
Keyword(s):  
Copy Number ◽  
Gene Dosage ◽  
Spindle Pole Body ◽  
Direct Interaction ◽  
Spindle Pole ◽  
High Copy Number ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Recessive Mutations ◽  
Organizing Center

Abstract KAR1 encodes an essential component of the yeast spindle pole body (SPB) that is required for karyogamy and SPB duplication. A temperature-sensitive mutation, kar1-delta 17, mapped to a region required for SPB duplication and for localization to the SPB. To identify interacting SPB proteins, we isolated 13 dominant mutations and 3 high copy number plasmids that suppressed the temperature sensitivity of kar1-delta 17. Eleven extragenic suppressor mutations mapped to two linkage groups, DSK1 and DSK2. The extragenic suppressors were specific for SPB duplication and did not suppress karyogamy-defective alleles. The major class, DSK1, consisted of mutations in CDC31. CDC31 is required for SPB duplication and encodes a calmodulin-like protein that is most closely related to caltractin/centrin, a protein associated with the Chlamydomonas basal body. The high copy number suppressor plasmids contained the wild-type CDC31 gene. One CDC31 suppressor allele conferred a temperature-sensitive defect in SPB duplication, which was counter-suppressed by recessive mutations in KAR1. In spite of the evidence for a direct interaction, the strongest CDC31 alleles, as well as both DSK2 alleles, suppressed a complete deletion of KAR1. However, the CDC31 alleles also made the cell supersensitive to KAR1 gene dosage, arguing against a simple bypass mechanism of suppression. We propose a model in which Kar1p helps localize Cdc31p to the SPB and that Cdc31p then initiates SPB duplication via interaction with a downstream effector.

scholarly journals Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex recruitment to the microtubule organizing center, but not its assembly

2013 ◽  
Vol 24 (18) ◽  
pp. 2894-2906 ◽  
Author(s):  
Hirohisa Masuda ◽  
Risa Mori ◽  
Masashi Yukawa ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Microtubule Organization ◽  
Unique Role ◽  
Pole Body ◽  
Organizing Center ◽  
Core Components

γ-Tubulin plays a universal role in microtubule nucleation from microtubule organizing centers (MTOCs) such as the animal centrosome and fungal spindle pole body (SPB). γ-Tubulin functions as a multiprotein complex called the γ-tubulin complex (γ-TuC), consisting of GCP1–6 (GCP1 is γ-tubulin). In fungi and flies, it has been shown that GCP1–3 are core components, as they are indispensable for γ-TuC complex assembly and cell division, whereas the other three GCPs are not. Recently a novel conserved component, MOZART1, was identified in humans and plants, but its precise functions remain to be determined. In this paper, we characterize the fission yeast homologue Mzt1, showing that it is essential for cell viability. Mzt1 is present in approximately equal stoichiometry with Alp4/GCP2 and localizes to all the MTOCs, including the SPB and interphase and equatorial MTOCs. Temperature-sensitive mzt1 mutants display varying degrees of compromised microtubule organization, exhibiting multiple defects during both interphase and mitosis. Mzt1 is required for γ-TuC recruitment, but not sufficient to localize to the SPB, which depends on γ-TuC integrity. Intriguingly, the core γ-TuC assembles in the absence of Mzt1. Mzt1 therefore plays a unique role within the γ-TuC components in attachment of this complex to the major MTOC site.

scholarly journals A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-Tubulin Complex

1998 ◽  
Vol 9 (8) ◽  
pp. 2201-2216 ◽  
Author(s):  
Thu Nguyen ◽  
Dani B.N. Vinh ◽  
Douglas K. Crawford ◽  
Trisha N. Davis
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Amino Terminus ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Synthetic Lethal ◽  
Lethal Phenotype ◽  
Amino Terminal ◽  
N Terminus ◽  
Two Hybrid

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110–221 allele, which encodes mutations in the amino terminus. The screen identified mutations inSPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98–63allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, thespc97 alleles are synthetic lethal withspc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98–63 spc110–221 cells is caused by the failure of Spc98–63p to interact with Spc110–221p. In contrast, the lethal phenotype in spc97–62 spc110–221 cells can be attributed to a decreased interaction between Spc97–62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.

scholarly journals Mobility, Microtubule Nucleation and Structure of Microtubule-organizing Centers in Multinucleated Hyphae ofAshbya gossypii

2010 ◽  
Vol 21 (1) ◽  
pp. 18-28 ◽  
Author(s):  
Claudia Lang ◽  
Sandrine Grava ◽  
Tineke van den Hoorn ◽  
Rhonda Trimble ◽  
Peter Philippsen ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Hyphal Growth ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
Microtubule Nucleation ◽  
Cytoplasmic Microtubule ◽  
Organizing Center ◽  
Cytoplasmic Side ◽  
Independent Mode

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 μm/min), rotations (up to 180° in 30 s), and long-range nuclear bypassing (up to 9 μm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis–oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

scholarly journals A Fourth Component of the Fission Yeast γ-Tubulin Complex, Alp16, Is Required for Cytoplasmic Microtubule Integrity and Becomes Indispensable When γ-Tubulin Function Is Compromised

2002 ◽  
Vol 13 (7) ◽  
pp. 2360-2373 ◽  
Author(s):  
Akiko Fujita ◽  
Leah Vardy ◽  
Miguel Angel Garcia ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Microtubule ◽  
Multicopy Suppressor ◽  
Large Complex ◽  
Synthetically Lethal ◽  
And Function

γ-Tubulin functions as a multiprotein complex, called the γ-tubulin complex (γ-TuC), and composes the microtubule organizing center (MTOC). Fission yeast Alp4 and Alp6 are homologues of two conserved γ-TuC proteins, hGCP2 and hGCP3, respectively. We isolated a novel gene, alp16 + , as a multicopy suppressor of temperature-sensitive alp6-719mutants. alp16 + encodes a 759-amino-acid protein with two conserved regions found in all other members of γ-TuC components. In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210. Gene disruption shows that alp16 + is not essential for cell viability. However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip. Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225,alp4-1891, or alp6-719 mutants. Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6. Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase. Alp16 coimmunoprecipitates with γ-tubulin and cosediments with the γ-TuC in a large complex (>20 S). Alp16 is, however, not required for the formation of this large complex. We discuss evolutional conservation and divergence of structure and function of the γ-TuC between yeast and higher eukaryotes.

scholarly journals Brr6 drives the Schizosaccharomyces pombe spindle pole body nuclear envelope insertion/extrusion cycle

2011 ◽  
Vol 195 (3) ◽  
pp. 467-484 ◽  
Author(s):  
Tiina Tamm ◽  
Agnes Grallert ◽  
Emily P.S. Grossman ◽  
Isabel Alvarez-Tabares ◽  
Frances E. Stevens ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Microtubule Organizing Center ◽  
Pole Body ◽  
Cytoplasmic Face ◽  
Organizing Center ◽  
Nuclear Component ◽  
Anaphase B

The fission yeast interphase spindle pole body (SPB) is a bipartite structure in which a bulky cytoplasmic domain is separated from a nuclear component by the nuclear envelope. During mitosis, the SPB is incorporated into a fenestra that forms within the envelope during mitotic commitment. Closure of this fenestra during anaphase B/mitotic exit returns the cytoplasmic component to the cytoplasmic face of an intact interphase nuclear envelope. Here we show that Brr6 is transiently recruited to SPBs at both SPB insertion and extrusion. Brr6 is required for both SPB insertion and nuclear envelope integrity during anaphase B/mitotic exit. Genetic interactions with apq12 and defective sterol assimilation suggest that Brr6 may alter envelope composition at SPBs to promote SPB insertion and extrusion. The restriction of the Brr6 domain to eukaryotes that use a polar fenestra in an otherwise closed mitosis suggests a conserved role in fenestration to enable a single microtubule organizing center to nucleate both cytoplasmic and nuclear microtubules on opposing sides of the nuclear envelope.

scholarly journals Localization of Core Spindle Pole Body (SPB) Components during SPB Duplication in Saccharomyces cerevisiae

1999 ◽  
Vol 145 (4) ◽  
pp. 809-823 ◽  
Author(s):  
Ian R. Adams ◽  
John V. Kilmartin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Large Plaque ◽  
Temperature Sensitive Mutants ◽  
Pole Body ◽  
Cytoplasmic Face ◽  
Preceding Cell

We have examined the process of spindle pole body (SPB) duplication in Saccharomyces cerevisiae by electron microscopy and found several stages. These include the assembly, probably from the satellite, of a large plaque-like structure, the duplication plaque, on the cytoplasmic face of the half-bridge and its insertion into the nuclear envelope. We analyzed the role of the main SPB components in the formation of these structures by identifying them from an SPB core fraction by mass spectrometry. Temperature-sensitive mutants for two of the components, Spc29p and Nud1p, were prepared to partly define their function. The composition of two of the intermediates in SPB duplication, the satellite and the duplication plaque, was examined by immunoelectron microscopy. Both contain cytoplasmic SPB components showing that duplication has already been partly achieved by the end of the preceding cell cycle when the satellite is formed. We show that by overexpression of SPB components the structure of the satellite can be changed and SPB duplication inhibited by disrupting the attachment of the plaque-like intermediate to the half-bridge. We present a model for SPB duplication where binding of SPB components to either end of the bridge structure ensures two separate SPBs.

Sign in / Sign up

Export Citation Format

Share Document