Stu2p binds tubulin and undergoes an open-to-closed conformational change

Al-Bassam, Jawdat; Breugel, M. van; Harrison, Stephen C.; Hyman, Anthony

doi:10.1083/jcb.200511010

Cited by 116 publications

(229 citation statements)

References 49 publications

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“…We asked whether Stu2⌬N localization is similar to endogenous Stu2 by tagging Stu2⌬N with venus fluorescent protein (VFP) (which still retains its spc24-9 HU rescue activity) and endogenous Stu2 with cyan fluorescent protein (CFP). Indeed, we found that Stu2⌬N-VFP localization overlapped with Stu2-CFP in both wild-type and spc24-9 mutants in log phase or HUtreated cells, consistent with previous data, demonstrating that Stu2⌬TOG1-GFP still binds MT plus ends (Supplemental Figure 2; Al-Bassam et al, 2006). To test whether depletion of Stu2 activity by using another mutant form of Stu2 is also capable of preventing spindle expansion in spc24-9 cells, we combined spc24-9 with the stu2-10 temperature-sensitive (Ts) mutation (Severin et al, 2001) and tested the double mutant for separation of nuclei upon HU treatment.…”

Section: Hcs Rescue Occurs Through Restraining Spindle Expansionsupporting

confidence: 78%

“…We propose that overexpression of STU2⌬N rescues spc24-9 HU lethality by restraining MT dynamics induced by mislocalization of Stu2. STU2⌬N lacks the N-terminal TOG1 domain that binds tubulin heterodimers but retains the TOG2 domain that binds MT plus ends (Al-Bassam et al, 2006). Previous studies have shown that Stu2 lacking its TOG1 domain binds MT plus ends but cannot promote plus-end MT growth, suggesting that STU2⌬N inhibits spc24-9 HU spindle expansion via the same mechanism (Al-Bassam et al, 2006).…”

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confidence: 99%

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Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled

McQueen

Cuschieri²,

Vogel

et al. 2007

MBoC

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The kinetochore, a protein complex that links chromosomes to microtubules (MTs), is required to prevent spindle expansion during S phase in budding yeast, but the mechanism of how the kinetochore maintains integrity of the bipolar spindle before mitosis is not well understood. Here, we demonstrate that a mutation of Spc24, a component of the conserved Ndc80 kinetochore complex, causes lethality when cells are exposed to the DNA replication inhibitor hydroxyurea (HU) due to premature spindle expansion and segregation of incompletely replicated DNA. Overexpression of Stu1, a CLASP-related MT-associated protein or a truncated form of the XMAP215 orthologue Stu2 rescues spc24-9 HU lethality and prevents spindle expansion. Truncated Stu2 likely acts in a dominant-negative manner, because overexpression of full-length STU2 does not rescue spc24-9 HU lethality, and spindle expansion in spc24-9 HU-treated cells requires active Stu2. Stu1 and Stu2 localize to the kinetochore early in the cell cycle and Stu2 kinetochore localization depends on Spc24. We propose that mislocalization of Stu2 results in premature spindle expansion in S phase stalled spc24-9 mutants. Identifying factors that restrain spindle expansion upon inhibition of DNA replication is likely applicable to the mechanism by which spindle elongation is regulated during a normal cell cycle. INTRODUCTIONPreserving the integrity of the genome is a fundamental requirement for eukaryotic cell viability. DNA replication must be completed before segregation of the chromosomes to prevent the transmission of partially replicated chromosomes to daughter cells. In the budding yeast Saccharomyces cerevisiae, cells undergo a closed mitosis and the microtubule (MT) organizing centers, or spindle pole bodies (SPBs), are imbedded in the nuclear envelope. SPB duplication begins at the end of anaphase and spindle formation begins during S phase when duplicated SPBs separate from each other (Adams and Kilmartin, 1999;Jaspersen and Winey, 2004). Because chromosomes remain attached to kinetochore MTs throughout the cell cycle, spindle expansion must be restrained until all 16 chromosomes have duplicated and kinetochores on sister chromatids have formed bipolar MT attachments. When DNA replication is stalled by hydroxyurea (HU) treatment, cells arrest with a large bud, an undivided nucleus positioned at the mother-bud neck and a short bipolar spindle (Allen et al., 1994). Maintaining a short spindle is crucial for cell survival during HU-induced arrest, which activates the DNA replication checkpoint effectors Mec1 and Rad53 (Kolodner et al., 2002). When mec1 and rad53 mutants are treated with HU, the DNA replication checkpoint is not activated, and, as a result, replication forks are not stabilized, the spindle expands, and unequal division of incompletely replicated nuclear material occurs-all of these events contribute to cell lethality (Allen et al., 1994;Weinert et al., 1994;Lopes et al., 2001).In human cells, stalled replication forks activate the ataxia telangiectasia mutat...

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Section: Hcs Rescue Occurs Through Restraining Spindle Expansionsupporting

confidence: 78%

mentioning

confidence: 99%

Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled

McQueen

Cuschieri²,

Vogel

et al. 2007

MBoC

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“…Acquired or inherited mutation in genes of the spindle checkpoint have been linked to several cancers (30)(31)(32)(33). For example, insertions and point mutations in both the kinetochore localization domains and the kinase domains of Bub1 and BubR1 have been found in leukemias and lymphomas (34)(35)(36).…”

Section: Discussionmentioning

confidence: 99%

“…Sedimentation equilibrium measurements of Mad3 and Mad3-Bub3 were made at 4°C in an analytical ultracentrifuge (Optima XLA; Beckman Coulter, Fullerton, CA) as described in ref. 30. Mad3 was prepared in 25 mM Bis-Tris-Pro, pH 6.5/250 mM NaCl/1 mM 2-mercaptoethanol, and Mad3-Bub3 was prepared in 10 mM Tris, pH 8.0/250 mM NaCl/1 mM 2-mercaptoethanol.…”

Section: Expression and Purification Of Saccharomyces Cerevisiae Checmentioning

confidence: 99%

Structural analysis of Bub3 interactions in the mitotic spindle checkpoint

Larsen

Al-Bassam

Wei

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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130

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The Mad3/BubR1, Mad2, Bub1, and Bub3 proteins are gatekeepers for the transition from metaphase to anaphase. Mad3 from Saccharomyces cerevisiae has homology to Bub1 but lacks a corresponding C-terminal kinase domain. Mad3 forms a stable heterodimer with Bub3. Negative-stain electron microscopy shows that Mad3 is an extended molecule (Ϸ200 Å long), whereas Bub3 is globular. The Gle2-binding-sequence (GLEBS) motifs found in Mad3 and Bub1 are necessary and sufficient for interaction with Bub3. The calorimetrically determined dissociation constants for GLEBS-motif peptides and Bub3 are Ϸ5 M. Crystal structures of these peptides with Bub3 show that the interactions for Mad3 and Bub1 are similar and mutually exclusive. In both structures, the GLEBS peptide snakes along the top surface of the ␤-propeller, forming an extensive interface. Mutations in either protein that disrupt the interface cause checkpoint deficiency and chromosome instability. We propose that the structure imposed on the GLEBS segment by its association with Bub3 enables recruitment to unattached kinetochores.␤-propeller ͉ crystal structures ͉ Rae1/Nup98 ͉ GLEBS motif

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“…6). This phenotype is not common, since most of the mutants of factors aVecting microtubules such as Bim1, Bik1 and Stu2 are more sensitive to such drugs (Al-Bassam et al 2006;Berlin et al 1990;Schwartz et al 1997;Winsor and Schiebel 1997).…”

Section: Discussionmentioning

confidence: 99%

The evolutionary conserved BER1 gene is involved in microtubule stability in yeast

et al. 2007

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In yeast, microtubules are dynamic Wlaments necessary for spindle and nucleus positioning, as well as for proper chromosome segregation. We identify a function for the yeast gene BER1 (Benomyl REsistant 1) in microtubule stability. BER1 belongs to an evolutionary conserved gene family whose founding member Sensitivity to Red light Reduced is involved in red-light perception and circadian rhythms in Arabidopsis. Here, we present data showing that the ber1 mutant is aVected in microtubule stability, particularly in presence of microtubuledepolymerising drugs. The pattern of synthetic lethal interactions obtained with the ber1 mutant suggests that Ber1 may function in N-terminal protein acetylation. Our work thus suggests that microtubule stability might be regulated through this post-translational modiWcation on yetto-be determined proteins.

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Stu2p binds tubulin and undergoes an open-to-closed conformational change

Cited by 116 publications

References 49 publications

Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled

Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled

Structural analysis of Bub3 interactions in the mitotic spindle checkpoint

The evolutionary conserved BER1 gene is involved in microtubule stability in yeast

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