We have developed a biochemical screen for the identification of kinesin- related proteins (KRPs) in their natural host cells and the subsequent purification of these KRPS as native, functional multimeric complexes. The screen involves immunoblotting with pan-kinesin peptide antibodies that recognize several presumptive KRPs in cytosolic extracts; the antibodies have been used so far to monitor the purification of two bona fide kinesin- related motor protein complexes. These two KRPs were purified via AMPPNP- induced microtubule affinity binding, ATP-induced elution from AMPPNP microtubules, gel filtration fractionation, and sucrose density gradient centrifugation. KRP(85/95) from sea urchin (Strongylocentrotus purpuratus) eggs behaves as a heterotrimeric complex of 85-, 95-, and 115- kDa subunits that moves toward the plus ends of microtubule tracks at approximately 0.4 μm/s. KRP(130) from fruitfly (Drosophila melanogaster) embryos behaves as a homotetrameric complex of four 130-kDa subunits that moves toward the plus ends of microtubule tracks at approximately 0.04 μm/s. To our knowledge, KRP(85/95) and KRP(130) are the only KRPS to have been purified from native tissue as functional multimeric motor complexes.
|Original language||English (US)|
|Issue number||4 SUPPL.|
|State||Published - Jan 1 1995|
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