Yu H, Nicchitta C V, Kumar J, Becker M, Toyoshima I, Sheetz M P
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Mol Biol Cell. 1995 Feb;6(2):171-83. doi: 10.1091/mbc.6.2.171.
Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.
驱动素结合蛋白是一种与驱动蛋白结合的蛋白质（丰岛等人，1992年），是基于驱动蛋白的运动所必需的（库马尔等人，1995年）。通过用一组单克隆抗体进行免疫筛选，从胚胎鸡脑cDNA文库中分离出一个含有4.7千碱基插入片段的驱动素结合蛋白cDNA克隆。该cDNA包含一个1364个氨基酸的开放阅读框，编码一个156 kDa的蛋白质。全长cDNA的细菌表达产物与纯化的驱动蛋白结合。在CV-1细胞中的瞬时表达产生了依赖于N端结构域的内质网分布。对预测的氨基酸序列分析表明，N端附近有一段28个氨基酸的高度疏水序列，以及预测的α螺旋卷曲螺旋的很大一部分（326-1248）。含有N端疏水区域的30 kDa片段通过无细胞体外翻译产生，并发现与犬胰腺粗面微粒体组装。未观察到N端的切割，证实了其作为潜在跨膜结构域的作用。因此，驱动素结合蛋白cDNA编码一种面向细胞质的整合膜蛋白，该蛋白与驱动蛋白结合，可能是一种卷曲螺旋二聚体。
