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Dynactin is required for bidirectional organelle transport.

Deacon SW, Serpinskaya AS, Vaughan PS, Lopez Fanarraga M, Vernos I, Vaughan KT, Gelfand VI - J. Cell Biol. (2003)

Bottom Line: Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II-associated protein (XKAP), binds directly to the p150Glued subunit of dynactin.This interaction occurs through aa 530-793 of XKAP and aa 600-811 of p150Glued.These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801.

ABSTRACT
Kinesin II is a heterotrimeric plus end-directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II-mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II-associated protein (XKAP), binds directly to the p150Glued subunit of dynactin. This interaction occurs through aa 530-793 of XKAP and aa 600-811 of p150Glued. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.

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Kinesin II and dynein compete for binding to dynactin. (A) Polyclonal antibodies against DIC and the 95-kD subunit of kinesin II are each able to pull down p150 from melanophore extracts, but they do not pull down each other. Antibodies used for immunoprep are shown at the top. Antibodies used to probe the blot are indicated on the sides. (B) The ability of p150 to bind to purified DIC via blot overlay diminishes in the presence of increasing amounts of C-XKAP but not N-XKAP. Purified DIC was overlayed with myc- p150Glued 600–811 alone or in the presence of a 1:1 or 1:2 molar ratio of C-XKAP or N-XKAP. Blots were probed with an anti-myc antibody.
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fig3: Kinesin II and dynein compete for binding to dynactin. (A) Polyclonal antibodies against DIC and the 95-kD subunit of kinesin II are each able to pull down p150 from melanophore extracts, but they do not pull down each other. Antibodies used for immunoprep are shown at the top. Antibodies used to probe the blot are indicated on the sides. (B) The ability of p150 to bind to purified DIC via blot overlay diminishes in the presence of increasing amounts of C-XKAP but not N-XKAP. Purified DIC was overlayed with myc- p150Glued 600–811 alone or in the presence of a 1:1 or 1:2 molar ratio of C-XKAP or N-XKAP. Blots were probed with an anti-myc antibody.

Mentions: The region of p150Glued implicated in kinesin II binding was also sufficient to bind the cytoplasmic dynein intermediate chain. This raised the question of whether dynactin could bind kinesin II and dynein simultaneously. To assess this possibility, immunoprecipitations were performed using polyclonal antibodies against the 95-kD subunit of kinesin II and DIC (Fig. 3 A). Both kinesin II and DIC were able to pull out p150Glued. However, no kinesin II was detected in antidynein immunoprecipitates and vice versa, suggesting that the two motors cannot bind to p150Glued at the same time.


Dynactin is required for bidirectional organelle transport.

Deacon SW, Serpinskaya AS, Vaughan PS, Lopez Fanarraga M, Vernos I, Vaughan KT, Gelfand VI - J. Cell Biol. (2003)

Kinesin II and dynein compete for binding to dynactin. (A) Polyclonal antibodies against DIC and the 95-kD subunit of kinesin II are each able to pull down p150 from melanophore extracts, but they do not pull down each other. Antibodies used for immunoprep are shown at the top. Antibodies used to probe the blot are indicated on the sides. (B) The ability of p150 to bind to purified DIC via blot overlay diminishes in the presence of increasing amounts of C-XKAP but not N-XKAP. Purified DIC was overlayed with myc- p150Glued 600–811 alone or in the presence of a 1:1 or 1:2 molar ratio of C-XKAP or N-XKAP. Blots were probed with an anti-myc antibody.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172679&req=5

fig3: Kinesin II and dynein compete for binding to dynactin. (A) Polyclonal antibodies against DIC and the 95-kD subunit of kinesin II are each able to pull down p150 from melanophore extracts, but they do not pull down each other. Antibodies used for immunoprep are shown at the top. Antibodies used to probe the blot are indicated on the sides. (B) The ability of p150 to bind to purified DIC via blot overlay diminishes in the presence of increasing amounts of C-XKAP but not N-XKAP. Purified DIC was overlayed with myc- p150Glued 600–811 alone or in the presence of a 1:1 or 1:2 molar ratio of C-XKAP or N-XKAP. Blots were probed with an anti-myc antibody.
Mentions: The region of p150Glued implicated in kinesin II binding was also sufficient to bind the cytoplasmic dynein intermediate chain. This raised the question of whether dynactin could bind kinesin II and dynein simultaneously. To assess this possibility, immunoprecipitations were performed using polyclonal antibodies against the 95-kD subunit of kinesin II and DIC (Fig. 3 A). Both kinesin II and DIC were able to pull out p150Glued. However, no kinesin II was detected in antidynein immunoprecipitates and vice versa, suggesting that the two motors cannot bind to p150Glued at the same time.

Bottom Line: Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II-associated protein (XKAP), binds directly to the p150Glued subunit of dynactin.This interaction occurs through aa 530-793 of XKAP and aa 600-811 of p150Glued.These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801.

ABSTRACT
Kinesin II is a heterotrimeric plus end-directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II-mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II-associated protein (XKAP), binds directly to the p150Glued subunit of dynactin. This interaction occurs through aa 530-793 of XKAP and aa 600-811 of p150Glued. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.

Show MeSH