Yu H, Toyoshima I, Steuer E R, Sheetz M P
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1992 Oct 5;267(28):20457-64.
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.
细胞细胞器沿着极性微管以定向方式移动是由驱动蛋白驱动的，驱动蛋白包括驱动蛋白和细胞质动力蛋白。本文研究了这些蛋白质与来自鸡胚脑的微粒体部分的结合情况。两种驱动蛋白都表现出对囊泡的饱和结合，并且囊泡膜蛋白的蛋白水解会消除结合。在有或没有1 mM ATP的情况下，驱动蛋白的最大结合量分别为每毫克囊泡蛋白12±1.7和43±2 pmol。在有或没有ATP的情况下，细胞质动力蛋白的最大结合量分别为每毫克囊泡蛋白55±3.8和73±3.7 pmol。这些值对应于直径为100 nm的每个囊泡有1 - 6个位点。不可水解的ATP类似物腺苷 - 5'-亚氨二磷酸（AMP - PNP）抑制驱动蛋白与囊泡的结合，但增加驱动蛋白与微管的结合。针对驱动蛋白轻链的抗体也抑制囊泡与驱动蛋白的结合。在没有ATP但有ATP时未观察到两种驱动蛋白之间的结合竞争。我们认为，在有ATP存在时，这些细胞器上存在驱动蛋白和细胞质动力蛋白的两个可区分的结合位点，而在没有ATP时存在一个共享位点。
