Abstract
Cytoplasmic dynein and kinesin I are both unidirectional intracellular motors. Dynein moves cargo toward the cell center, and kinesin moves cargo toward the cell periphery. There is growing evidence that bi-directional motility is regulated in the cell, potentially through direct interactions between oppositely oriented motors. We have identified a direct interaction between cytoplasmic dynein and kinesin I. Using the yeast two-hybrid assay and affinity chromatography, we demonstrate that the intermediate chain of dynein binds to kinesin light chains 1 and 2. The interaction is both direct and specific. Co-immunoprecipitation experiments demonstrate an interaction between endogenous proteins in rat brain cytosol. Double-label immunocytochemistry reveals a partial co-localization of vesicle-associated motor proteins. Together these observations suggest that soluble motors can interact, potentially allowing kinesin I to actively localize dynein to cellular sites of function. There is also a vesicle population with both dynein and kinesin I bound that may be capable of bi-directional motility along cellular microtubules.
Highlights
Intracellular transport along microtubules is driven by the motors kinesin and cytoplasmic dynein
We performed a yeast two-hybrid screen for proteins interacting with dynein intermediate chain (DIC) and isolated a clone encoding TPR1, a human protein with three tetratricopeptide repeats (TPR)
Association of vesicles with microtubules was more pronounced in the presence of AMP-PNP, and the addition of MgATP induced significant dissociation. These localization studies are consistent with results from immunocytochemistry, indicating that the most robust association between dynein and kinesin occurs in the soluble pool of motors, there is a population of vesicles with both motors bound. These observations identify a direct interaction between cytoplasmic dynein and kinesin, mediated by kinesin light chains
Summary
Yeast Two-hybrid Interaction Screen—We used the LexA yeast twohybrid system to screen an oligo(T)-primed human fetal brain library (Stratagene) for proteins interacting with DIC. Binding and domain mapping studies between kinesin and dynein were performed by subcloning full-length or partial sequences, as noted, of DIC, KLC1, and KLC2 into either the pB42Ad or pJK202 vectors (Stratagene) and scoring for growth on medium lacking adenine in comparison with the appropriate empty vector, as previously described [4]. Purified recombinant DIC or rat brain cytosolic extracts were fractionated over affinity columns generated from either GST fusion proteins or GST alone bound to glutathione-Sepharose, followed by elution of bound proteins with glutathione [4]. The binding of kinesin to DIC was examined by fractionating either purified recombinant KLCs, in vitro translated truncation constructs, or rat brain cytosol over affinity
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