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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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Recombinant, baculovirus-derived Stu2p and endogenous Stu2p show similar hydrodynamic properties. (A) Coomassie blue–stained SDS-polyacrylamide gel showing ∼7.5 μg of purified, baculovirus-derived Stu2p. (B) Determination of the Stokes radius of baculovirus-derived and endogenous Stu2p in yeast extracts using gel filtration on a Superose 6 column. The fractions with recombinant Stu2p (top) or yeast extract (bottom) were separated by SDS-PAGE, and Stu2p was detected by Western blotting using a polyclonal Stu2p antibody. The corresponding positions of selected markers of known Stokes radius are indicated by arrows. (C) Determination of the S value of recombinant and endogenous Stu2p in yeast extracts. Recombinant Stu2p and endogenous Stu2p in yeast extracts were spun through a linear 5–40% sucrose gradient. Fractions were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody. (Top) Western blot for recombinant Stu2p; (bottom) Western blot for endogenous Stu2p. Corresponding positions of markers with known S value are indicated by arrows.
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fig1: Recombinant, baculovirus-derived Stu2p and endogenous Stu2p show similar hydrodynamic properties. (A) Coomassie blue–stained SDS-polyacrylamide gel showing ∼7.5 μg of purified, baculovirus-derived Stu2p. (B) Determination of the Stokes radius of baculovirus-derived and endogenous Stu2p in yeast extracts using gel filtration on a Superose 6 column. The fractions with recombinant Stu2p (top) or yeast extract (bottom) were separated by SDS-PAGE, and Stu2p was detected by Western blotting using a polyclonal Stu2p antibody. The corresponding positions of selected markers of known Stokes radius are indicated by arrows. (C) Determination of the S value of recombinant and endogenous Stu2p in yeast extracts. Recombinant Stu2p and endogenous Stu2p in yeast extracts were spun through a linear 5–40% sucrose gradient. Fractions were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody. (Top) Western blot for recombinant Stu2p; (bottom) Western blot for endogenous Stu2p. Corresponding positions of markers with known S value are indicated by arrows.

Mentions: To investigate the biochemical activities of Stu2p, we sought to make pure recombinant protein. Stu2p has previously been expressed in reticulate lysates (Wang and Huffaker, 1997), but the amounts obtained were too small for tubulin-dependent assays. Although Escherichia coli–expressed Stu2p is apparently soluble, it aggregated in the presence of microtubules (unpublished data). Therefore, we expressed and purified Stu2p using recombinant baculovirus. Typically, a 500-ml insect cell culture yielded 4 mg protein that was over 90% pure (Fig. 1 A).


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)

Recombinant, baculovirus-derived Stu2p and endogenous Stu2p show similar hydrodynamic properties. (A) Coomassie blue–stained SDS-polyacrylamide gel showing ∼7.5 μg of purified, baculovirus-derived Stu2p. (B) Determination of the Stokes radius of baculovirus-derived and endogenous Stu2p in yeast extracts using gel filtration on a Superose 6 column. The fractions with recombinant Stu2p (top) or yeast extract (bottom) were separated by SDS-PAGE, and Stu2p was detected by Western blotting using a polyclonal Stu2p antibody. The corresponding positions of selected markers of known Stokes radius are indicated by arrows. (C) Determination of the S value of recombinant and endogenous Stu2p in yeast extracts. Recombinant Stu2p and endogenous Stu2p in yeast extracts were spun through a linear 5–40% sucrose gradient. Fractions were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody. (Top) Western blot for recombinant Stu2p; (bottom) Western blot for endogenous Stu2p. Corresponding positions of markers with known S value are indicated by arrows.
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Related In: Results  -  Collection

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fig1: Recombinant, baculovirus-derived Stu2p and endogenous Stu2p show similar hydrodynamic properties. (A) Coomassie blue–stained SDS-polyacrylamide gel showing ∼7.5 μg of purified, baculovirus-derived Stu2p. (B) Determination of the Stokes radius of baculovirus-derived and endogenous Stu2p in yeast extracts using gel filtration on a Superose 6 column. The fractions with recombinant Stu2p (top) or yeast extract (bottom) were separated by SDS-PAGE, and Stu2p was detected by Western blotting using a polyclonal Stu2p antibody. The corresponding positions of selected markers of known Stokes radius are indicated by arrows. (C) Determination of the S value of recombinant and endogenous Stu2p in yeast extracts. Recombinant Stu2p and endogenous Stu2p in yeast extracts were spun through a linear 5–40% sucrose gradient. Fractions were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody. (Top) Western blot for recombinant Stu2p; (bottom) Western blot for endogenous Stu2p. Corresponding positions of markers with known S value are indicated by arrows.
Mentions: To investigate the biochemical activities of Stu2p, we sought to make pure recombinant protein. Stu2p has previously been expressed in reticulate lysates (Wang and Huffaker, 1997), but the amounts obtained were too small for tubulin-dependent assays. Although Escherichia coli–expressed Stu2p is apparently soluble, it aggregated in the presence of microtubules (unpublished data). Therefore, we expressed and purified Stu2p using recombinant baculovirus. Typically, a 500-ml insect cell culture yielded 4 mg protein that was over 90% pure (Fig. 1 A).

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