scholarly journals The fork head transcription factor Hcm1p participates in the regulation of SPC110, which encodes the calmodulin-binding protein in the yeast spindle pole body

1998 ◽  
Vol 1448 (2) ◽  
pp. 236-244 ◽  
Author(s):  
Gefeng Zhu ◽  
Trisha N. Davis
Keyword(s):  
Transcription Factor ◽  
Binding Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Calmodulin Binding ◽  
Fork Head ◽  
Pole Body

scholarly journals The Fission Yeast Homolog of the Human Transcription Factor EAP30 Blocks Meiotic Spindle Pole Body Amplification

2005 ◽  
Vol 9 (1) ◽  
pp. 63-73 ◽  
Author(s):  
Ye Jin ◽  
Joel J. Mancuso ◽  
Satoru Uzawa ◽  
Daniela Cronembold ◽  
W. Zacheus Cande
Keyword(s):  
Transcription Factor ◽  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Pole Body ◽  
Yeast Homolog ◽  
Human Transcription Factor

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (12) ◽  
pp. 7913-7924
Author(s):  
J R Geiser ◽  
H A Sundberg ◽  
B H Chang ◽  
E G Muller ◽  
T N Davis
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Binding Site ◽  
Hybrid System ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Amino Acid Residues ◽  
Calmodulin Binding ◽  
Pole Body ◽  
Two Hybrid

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.

scholarly journals Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents.

1996 ◽  
Vol 133 (1) ◽  
pp. 111-124 ◽  
Author(s):  
H A Sundberg ◽  
L Goetsch ◽  
B Byers ◽  
T N Davis
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Immunofluorescent Staining ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Nonpermissive Temperature ◽  
Carboxy Terminus ◽  
Calmodulin Binding ◽  
Pole Body ◽  
Mutant Cells

Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation. Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque. We created temperature-sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy. The temperature-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature-sensitive phenotype. Calmodulin overproduction suppresses the temperature sensitivity of spc110-220. Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature. Thus, calmodulin binding to Spc110-220p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality. Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles. Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from a SPB but serves as a center of microtubule organization. The IMO appears during nascent SPB formation and disappears after SPB separation. The IMO contains both the 90-kD and the mutant 110-kD SPB components. Our results suggest that disruption of the calmodulin Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation.

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

2000 ◽  
Vol 11 (9) ◽  
pp. 2949-2959 ◽  
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.

scholarly journals Regulation of spindle pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast

2014 ◽  
Vol 25 (18) ◽  
pp. 2735-2749 ◽  
Author(s):  
I-Ju Lee ◽  
Ning Wang ◽  
Wen Hu ◽  
Kersey Schott ◽  
Jürg Bähler ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Fission Yeast ◽  
Budding Yeast ◽  
Binding Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Centrosome Duplication ◽  
Pole Body ◽  
The Individual

Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly.

scholarly journals The Fission Yeast Homolog of the Human Transcription Factor EAP30 Blocks Meiotic Spindle Pole Body Amplification

2005 ◽  
Vol 9 (3) ◽  
pp. 439
Author(s):  
Ye Jin ◽  
Joel J. Mancuso ◽  
Satoru Uzawa ◽  
Daniela Cronembold ◽  
W. Zacheus Cande
Keyword(s):  
Transcription Factor ◽  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Pole Body ◽  
Yeast Homolog ◽  
Human Transcription Factor

scholarly journals The N-terminus of Sfi1 and yeast centrin Cdc31 provide the assembly site for a new spindle pole body

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Diana Rüthnick ◽  
Jlenia Vitale ◽  
Annett Neuner ◽  
Elmar Schiebel
Keyword(s):  
Cell Cycle ◽  
Binding Sites ◽  
Binding Protein ◽  
Spindle Pole Body ◽  
Inhibitory Effect ◽  
Spindle Pole ◽  
Satellite Formation ◽  
Pole Body ◽  
N Terminus ◽  
Assembly Site

The spindle pole body (SPB) provides microtubule-organizing functions in yeast and duplicates exactly once per cell cycle. The first step in SPB duplication is the half-bridge to bridge conversion via the antiparallel dimerization of the centrin (Cdc31)-binding protein Sfi1 in anaphase. The bridge, which is anchored to the old SPB on the proximal end, exposes free Sfi1 N-termini (N-Sfi1) at its distal end. These free N-Sfi1 promote in G1 the assembly of the daughter SPB (dSPB) in a yet unclear manner. This study shows that N-Sfi1 including the first three Cdc31 binding sites interacts with the SPB components Spc29 and Spc42, triggering the assembly of the dSPB. Cdc31 binding to N-Sfi1 promotes Spc29 recruitment and is essential for satellite formation. Furthermore, phosphorylation of N-Sfi1 has an inhibitory effect and delays dSPB biogenesis until G1. Taking these data together, we provide an understanding of the initial steps in SPB assembly and describe a new function of Cdc31 in the recruitment of dSPB components.

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