Purification of kinesin-related protein complexes from eggs and embryos.
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 micron/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 micron/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.
Cole
D G
DG
Section of Molecular and Cellular Biology, University of California at Davis 95616, USA.
Scholey
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D004331:Drosophila melanogaster; D004625:Embryo, Nonmammalian / Q000737:chemistry*; D005260:Female; D016547:Kinesins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008868:Microtubule Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010063:Ovum / Q000737:chemistry*; D011487:Protein Conformation; D012617:Sea Urchins
D008868:Microtubule Proteins; D016547:Kinesins