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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 the dynactin complex interact in melanophore extracts. (A) Kinesin II was precipitated from melanophore extracts with five different polyclonal (left) and one monoclonal (right) antibodies against the 95- and 85-kD subunits of kinesin II, respectively. Blots were probed with monoclonal anti-p150. Quantification of these blots shows that different kinesin II antibodies pull down 2–6% of the total p150 in the extract. (B) Dynactin was precipitated with polyclonal (left) or monoclonal (right) antibodies against p150. Blots were probed with monoclonal anti–kinesin II. Quantification of these blots shows that p150 antibodies pull down ∼1% of the total kinesin II in the extract. C indicates control samples precipitated either by normal rabbit IgG (left) or an unrelated monoclonal antibody (right).
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fig1: Kinesin II and the dynactin complex interact in melanophore extracts. (A) Kinesin II was precipitated from melanophore extracts with five different polyclonal (left) and one monoclonal (right) antibodies against the 95- and 85-kD subunits of kinesin II, respectively. Blots were probed with monoclonal anti-p150. Quantification of these blots shows that different kinesin II antibodies pull down 2–6% of the total p150 in the extract. (B) Dynactin was precipitated with polyclonal (left) or monoclonal (right) antibodies against p150. Blots were probed with monoclonal anti–kinesin II. Quantification of these blots shows that p150 antibodies pull down ∼1% of the total kinesin II in the extract. C indicates control samples precipitated either by normal rabbit IgG (left) or an unrelated monoclonal antibody (right).

Mentions: One explanation for these results is that both kinesin II and dynein share the same membrane receptor. To test this hypothesis, we performed immunoprecipitations of melanophore extracts using a monoclonal antibody raised against the 85-kD subunit of kinesin II (K2.4). Coimmunoprecipitation of dynactin with kinesin II was observed with this antibody but not control antibodies (Fig. 1 A). To exclude cross-reaction with dynactin as an explanation, we repeated this experiment using five different affinity-purified polyclonal antibodies against kinesin II. Each polyclonal antibody, but not control IgG, was able to pull down dynactin from melanophore extracts (Fig. 1 A). In these experiments, monoclonal and polyclonal antibodies against kinesin II pulled out 2–6% of the total dynactin pool. In the reverse experiment, both polyclonal and monoclonal antibodies to the p150Glued subunit of the dynactin complex pulled out ∼1% of the total kinesin II pool (Fig. 1 B). These results provided an indication that kinesin II and dynactin interact.


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 the dynactin complex interact in melanophore extracts. (A) Kinesin II was precipitated from melanophore extracts with five different polyclonal (left) and one monoclonal (right) antibodies against the 95- and 85-kD subunits of kinesin II, respectively. Blots were probed with monoclonal anti-p150. Quantification of these blots shows that different kinesin II antibodies pull down 2–6% of the total p150 in the extract. (B) Dynactin was precipitated with polyclonal (left) or monoclonal (right) antibodies against p150. Blots were probed with monoclonal anti–kinesin II. Quantification of these blots shows that p150 antibodies pull down ∼1% of the total kinesin II in the extract. C indicates control samples precipitated either by normal rabbit IgG (left) or an unrelated monoclonal antibody (right).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172679&req=5

fig1: Kinesin II and the dynactin complex interact in melanophore extracts. (A) Kinesin II was precipitated from melanophore extracts with five different polyclonal (left) and one monoclonal (right) antibodies against the 95- and 85-kD subunits of kinesin II, respectively. Blots were probed with monoclonal anti-p150. Quantification of these blots shows that different kinesin II antibodies pull down 2–6% of the total p150 in the extract. (B) Dynactin was precipitated with polyclonal (left) or monoclonal (right) antibodies against p150. Blots were probed with monoclonal anti–kinesin II. Quantification of these blots shows that p150 antibodies pull down ∼1% of the total kinesin II in the extract. C indicates control samples precipitated either by normal rabbit IgG (left) or an unrelated monoclonal antibody (right).
Mentions: One explanation for these results is that both kinesin II and dynein share the same membrane receptor. To test this hypothesis, we performed immunoprecipitations of melanophore extracts using a monoclonal antibody raised against the 85-kD subunit of kinesin II (K2.4). Coimmunoprecipitation of dynactin with kinesin II was observed with this antibody but not control antibodies (Fig. 1 A). To exclude cross-reaction with dynactin as an explanation, we repeated this experiment using five different affinity-purified polyclonal antibodies against kinesin II. Each polyclonal antibody, but not control IgG, was able to pull down dynactin from melanophore extracts (Fig. 1 A). In these experiments, monoclonal and polyclonal antibodies against kinesin II pulled out 2–6% of the total dynactin pool. In the reverse experiment, both polyclonal and monoclonal antibodies to the p150Glued subunit of the dynactin complex pulled out ∼1% of the total kinesin II pool (Fig. 1 B). These results provided an indication that kinesin II and dynactin interact.

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