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Stu2p, the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer.

van Breugel M, Drechsel D, Hyman A - J. Cell Biol. (2003)

Bottom Line: Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends.Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends.These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

ABSTRACT
The Dis1/XMAP215 family of microtubule-associated proteins conserved from yeast to mammals is essential for cell division. XMAP215, the Xenopus member of this family, has been shown to stabilize microtubules in vitro, but other members of this family have not been biochemically characterized. Here we investigate the properties of the Saccharomyces cerevisiae homologue Stu2p in vitro. Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends. Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends. These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.

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Stu2p does not strongly require the COOH terminus of β-tubulin for binding to microtubules. (A) Mock- or subtilisin- digested microtubules analyzed by SDS-PAGE and Coomassie blue staining (left) or by Western blotting (right) using a monoclonal α-tubulin antibody (top) or a monoclonal β-tubulin antibody whose epitope lies within the COOH terminus of β-tubulin (bottom). (B, top) Mock- and subtilisin-digested microtubules visualized by immunofluorescence. Bar, 5 μm. (B, bottom) Increasing amounts of mock- or subtilisin-digested microtubules were incubated with 18 nM Stu2p and bound separated from unbound Stu2p by centrifugation. Equivalent amounts of the supernatants (S) and the pellets (P) were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody (top), a monoclonal α-tubulin antibody (middle), or a monoclonal β-tubulin antibody whose epitope lies within the COOH terminus of β-tubulin (bottom). (C) Plot showing the percentage of Stu2p bound to mock- or subtilisin-digested microtubules at different microtubule concentrations.
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fig2: Stu2p does not strongly require the COOH terminus of β-tubulin for binding to microtubules. (A) Mock- or subtilisin- digested microtubules analyzed by SDS-PAGE and Coomassie blue staining (left) or by Western blotting (right) using a monoclonal α-tubulin antibody (top) or a monoclonal β-tubulin antibody whose epitope lies within the COOH terminus of β-tubulin (bottom). (B, top) Mock- and subtilisin-digested microtubules visualized by immunofluorescence. Bar, 5 μm. (B, bottom) Increasing amounts of mock- or subtilisin-digested microtubules were incubated with 18 nM Stu2p and bound separated from unbound Stu2p by centrifugation. Equivalent amounts of the supernatants (S) and the pellets (P) were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody (top), a monoclonal α-tubulin antibody (middle), or a monoclonal β-tubulin antibody whose epitope lies within the COOH terminus of β-tubulin (bottom). (C) Plot showing the percentage of Stu2p bound to mock- or subtilisin-digested microtubules at different microtubule concentrations.

Mentions: Many microtubule-associated proteins like MAP1, MAP2, and tau (Serrano et al., 1985), but not the human Stu2p homologue ch-TOG (Spittle et al., 2000), require the COOH-terminal tail of β-tubulin for microtubule binding. To determine the involvement of the β-tubulin COOH terminus in binding of Stu2p to microtubules, we made microtubules without the COOH terminus by limited subtilisin proteolysis of taxol-stabilized microtubules. Fig. 2 A shows a Coomassie-stained gel of mock-digested and digested microtubules and a Western blot probed with an antibody specific for the COOH terminus of β-tubulin. We determined the relative extent of binding of Stu2p to these microtubules by incubating increasing amounts of them with 18 nM Stu2p at room temperature. Bound Stu2p was separated from unbound Stu2p by centrifugation, supernatants and pellets were analyzed by Western blotting (Fig. 2 B), and the extent of binding was subsequently quantitated (Fig. 2 C). Over a wide concentration range of microtubules, Stu2p bound to a similar extent to the undigested and the digested microtubules. Thus, Stu2p, like its human homologue ch-TOG but unlike many other microtubule-associated proteins so far studied, does not seem to have a strong requirement of the COOH terminus of β-tubulin for microtubule binding.


Stu2p, the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer.

van Breugel M, Drechsel D, Hyman A - J. Cell Biol. (2003)

Stu2p does not strongly require the COOH terminus of β-tubulin for binding to microtubules. (A) Mock- or subtilisin- digested microtubules analyzed by SDS-PAGE and Coomassie blue staining (left) or by Western blotting (right) using a monoclonal α-tubulin antibody (top) or a monoclonal β-tubulin antibody whose epitope lies within the COOH terminus of β-tubulin (bottom). (B, top) Mock- and subtilisin-digested microtubules visualized by immunofluorescence. Bar, 5 μm. (B, bottom) Increasing amounts of mock- or subtilisin-digested microtubules were incubated with 18 nM Stu2p and bound separated from unbound Stu2p by centrifugation. Equivalent amounts of the supernatants (S) and the pellets (P) were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody (top), a monoclonal α-tubulin antibody (middle), or a monoclonal β-tubulin antibody whose epitope lies within the COOH terminus of β-tubulin (bottom). (C) Plot showing the percentage of Stu2p bound to mock- or subtilisin-digested microtubules at different microtubule concentrations.
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Related In: Results  -  Collection

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fig2: Stu2p does not strongly require the COOH terminus of β-tubulin for binding to microtubules. (A) Mock- or subtilisin- digested microtubules analyzed by SDS-PAGE and Coomassie blue staining (left) or by Western blotting (right) using a monoclonal α-tubulin antibody (top) or a monoclonal β-tubulin antibody whose epitope lies within the COOH terminus of β-tubulin (bottom). (B, top) Mock- and subtilisin-digested microtubules visualized by immunofluorescence. Bar, 5 μm. (B, bottom) Increasing amounts of mock- or subtilisin-digested microtubules were incubated with 18 nM Stu2p and bound separated from unbound Stu2p by centrifugation. Equivalent amounts of the supernatants (S) and the pellets (P) were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody (top), a monoclonal α-tubulin antibody (middle), or a monoclonal β-tubulin antibody whose epitope lies within the COOH terminus of β-tubulin (bottom). (C) Plot showing the percentage of Stu2p bound to mock- or subtilisin-digested microtubules at different microtubule concentrations.
Mentions: Many microtubule-associated proteins like MAP1, MAP2, and tau (Serrano et al., 1985), but not the human Stu2p homologue ch-TOG (Spittle et al., 2000), require the COOH-terminal tail of β-tubulin for microtubule binding. To determine the involvement of the β-tubulin COOH terminus in binding of Stu2p to microtubules, we made microtubules without the COOH terminus by limited subtilisin proteolysis of taxol-stabilized microtubules. Fig. 2 A shows a Coomassie-stained gel of mock-digested and digested microtubules and a Western blot probed with an antibody specific for the COOH terminus of β-tubulin. We determined the relative extent of binding of Stu2p to these microtubules by incubating increasing amounts of them with 18 nM Stu2p at room temperature. Bound Stu2p was separated from unbound Stu2p by centrifugation, supernatants and pellets were analyzed by Western blotting (Fig. 2 B), and the extent of binding was subsequently quantitated (Fig. 2 C). Over a wide concentration range of microtubules, Stu2p bound to a similar extent to the undigested and the digested microtubules. Thus, Stu2p, like its human homologue ch-TOG but unlike many other microtubule-associated proteins so far studied, does not seem to have a strong requirement of the COOH terminus of β-tubulin for microtubule binding.

Bottom Line: Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends.Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends.These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

ABSTRACT
The Dis1/XMAP215 family of microtubule-associated proteins conserved from yeast to mammals is essential for cell division. XMAP215, the Xenopus member of this family, has been shown to stabilize microtubules in vitro, but other members of this family have not been biochemically characterized. Here we investigate the properties of the Saccharomyces cerevisiae homologue Stu2p in vitro. Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends. Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends. These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.

Show MeSH