Georgatos S D, Marchesi V T
J Cell Biol. 1985 Jun;100(6):1955-61. doi: 10.1083/jcb.100.6.1955.
We have characterized the association of the intermediate filament protein, vimentin, with the plasma membrane, using radioiodinated lens vimentin and various preparations of human erythrocyte membrane vesicles. Inside-out membrane vesicles (IOVs), depleted of spectrin and actin, bind I125-vimentin in a saturable manner unlike resealed, right-side-out membranes which bind negligible amounts of vimentin in an unsaturable fashion. The binding of vimentin to IOVs is abolished by trypsin or acid treatment of the vesicles. Extraction of protein 4.1 or reconstitution of the membranes with purified spectrin do not basically affect the association. However, removal of ankyrin (band 2.1) significantly lowers the binding. Upon reconstitution of depleted vesicles with purified ankyrin, the vimentin binding function is restored. If ankyrin is added in excess the binding of vimentin to IOVs is quantitatively inhibited, whereas protein 4.1, the cytoplasmic fragment of band 3, band 6, band 4.5 (catalase), or bovine serum albumin do not influence it. Preincubation of the IOVs with a polyclonal anti-ankyrin antibody blocks 90% of the binding. Preimmune sera and antibodies against spectrin, protein 4.1, glycophorin A, and band 3 exhibit no effect. On the basis of these data, we propose that vimentin is able to associate specifically with the erythrocyte membrane skeleton and that ankyrin constitutes its major attachment site.
我们使用放射性碘化晶状体波形蛋白和各种人红细胞膜囊泡制剂，对中间丝蛋白波形蛋白与质膜的关联进行了表征。与重新封闭的、外翻的膜不同，耗尽血影蛋白和肌动蛋白的内翻膜囊泡（IOV）以饱和方式结合¹²⁵I-波形蛋白，而外翻膜以不饱和方式结合可忽略不计的波形蛋白量。波形蛋白与IOV的结合可通过用胰蛋白酶或酸处理囊泡来消除。提取蛋白4.1或用纯化的血影蛋白重建膜基本上不影响这种关联。然而，去除锚蛋白（带2.1）会显著降低结合。用纯化的锚蛋白重建耗尽的囊泡后，波形蛋白结合功能得以恢复。如果过量添加锚蛋白，波形蛋白与IOV的结合会被定量抑制，而蛋白4.1、带3的细胞质片段、带6、带4.5（过氧化氢酶）或牛血清白蛋白则不会影响它。用多克隆抗锚蛋白抗体预孵育IOV可阻断90%的结合。免疫前血清以及针对血影蛋白、蛋白4.1、血型糖蛋白A和带3的抗体均无作用。基于这些数据，我们提出波形蛋白能够与红细胞膜骨架特异性结合，并且锚蛋白构成其主要附着位点。
