Kinesin and cytoplasmic dynein binding to brain microsomes.
Movement of cellular organelles in a directional manner along polar microtubules is driven by the motor proteins, kinesin and cytoplasmic dynein. The binding of these proteins to a microsomal fraction from embryonic chicken brain is investigated here. Both motors exhibit saturation binding to the vesicles, and proteolysis of vesicle membrane proteins abolishes binding. The maximal binding for kinesin is 12 +/- 1.7 and 43 +/- 2 pmol per mg of vesicle protein with or without 1 mM ATP, respectively. The maximal binding for cytoplasmic dynein is 55 +/- 3.8 and 73 +/- 3.7 pmol per mg of vesicle protein with or without ATP, respectively. These values correspond to 1-6 sites per vesicle of 100-nm diameter. The nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate (AMP-PNP), inhibited kinesin binding to vesicles but increased kinesin binding to microtubules. An antibody to the kinesin light chain also inhibited vesicle binding to kinesin. In the absence but not presence of ATP, competition between the two motors for binding was observed. We suggest that there are two distinguishable binding sites for kinesin and cytoplasmic dynein on these organelles in the presence of ATP and a shared site in the absence of ATP.
Yu
H
H
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
Toyoshima
I
I
Steuer
E R
ER
Sheetz
M P
MP
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002642:Chick Embryo; D003593:Cytoplasm / Q000378:metabolism*; D004398:Dyneins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016547:Kinesins / Q000378:metabolism*; D008854:Microscopy, Electron; D008861:Microsomes / Q000378:metabolism* / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism
D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D004398:Dyneins; D016547:Kinesins