The nature of kinesin interactions with membrane-bound organelles and mecha
nisms for regulation of kinesin-based motility have both been surprisingly
difficult to define. Most kinesin is recovered in supernatants with standar
d protocols for purification of motor proteins, but kinesin recovered on me
mbrane-bound organelles is tightly bound. Partitioning of kinesin between v
esicle and cytosolic fractions is highly sensitive to buffer composition Ad
dition of either N-ethylmaleimide or EDTA to homogenization buffers signifi
cantly increased the fraction of kinesin bound to organelles. Given that an
antibody against kinesin light chain tandem repeats also releases kinesin
from vesicles, these observations indicated that specific cytoplasmic facto
rs may regulate kinesin release from membranes. Kinesin light tandem repeat
s contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluat
ed. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylm
aleimide-sensitive manner. Recombinant kinesin light chains inhibited kines
in release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may prov
ide a mechanism to regulate kinesin function by releasing kinesin from carg
o in specific subcellular domains, thereby effecting delivery of axonally t
ransported materials.