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Kinesin and cytoplasmic dynein binding to brain microsomes.

1992; Elsevier BV; Volume: 267; Issue: 28 Linguagem: Inglês

10.1016/s0021-9258(19)88724-0

1083-351X

Hanry Yu, Itaru Toyoshima, E. Steuer, M.P. Sheetz,

Mitochondrial Function and Pathology

Movement of cellular organelles ina 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 f 1.7 and 43 f 2 pmol per mg of vesicle protein with or without 1 m M ATP, respectively.The maximal binding for cytoplasmic dynein is 55 f 3.8 and 7 3 f 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.The axons and dendrites in brain must be supported by the active transport of organelles along microtubules (see reviews in Refs. 1-3).Kinesin and cytoplasmic dynein have been shown to drive the in vitro movement of vesicular organelles along microtubules toward the plus and minus ends of microtubules, respectively, and are believed to function similarly i n vivo (4-6).Using the in vitro reconstitution of motility as an assay, the minimal complex required for organelle motility has been defined as the motor protein, a vesicle binding site and an accessory factor (4). Once the correct motor protein binds to the appropriate vesicles and the motor complex is activated for motility, the vesicles will move in the correct direction along the microtubule.The exact nature of the interaction of the motors with the organelle surface is therefore critical for understanding the control of motility.The simplest method for controlling the direction of organelle movement would be to have a single binding site for both motor proteins that would only be activated for one of the